Biochemistry [6th ed.]

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Biochemistry [6th ed.]

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Lippinc o tt’s Illus trate d Re vie ws : Bio c he mis try S ixth Editio n

tahir99-VRG & vip.persianss.ir

Contents UNIT I: Protein Structure and Function Chapte r 1: Chapte r 2: Chapte r 3: Chapte r 4: Chapte r 5:

Amino Acids 1 S tructure of P rote ins 13 Globula r P rote ins 25 Fibrous P rote ins 43 Enzyme s 53

UNIT II: Bioenergetics and Carbohydrate Metabolism Chapte r 6: Chapte r 7: Chapte r 8: Chapte r 9: Chapte r 10: Chapte r 11: Chapte r 12: Chapte r 13: Chapte r 14:

Bioe ne rge tics a nd Oxida tive P hos phoryla tion 69 Introduction to Ca rbohydra te s 83 Introduction to Me ta bolis m a nd Glycolys is 91 Trica rboxylic Acid Cycle a nd P yruva te De hydroge na s e Comple x 109 Glucone oge ne s is 117 Glycoge n Me ta bolis m 125 Me ta bolis m of Monos a ccha ride s a nd Dis a ccha ride s 137 P e ntos e P hos pha te P a thwa y a nd Nicotina mide Ade nine Dinucle otide P hos pha te Glycos a minoglyca ns , P rote oglyca ns , a nd Glycoprote ins 157

145

UNIT III: Lipid Metabolism Chapte r 15: Chapte r 16: Chapte r 17: Chapte r 18:

Die ta ry Lipids Me ta bolis m 173 Fa tty Acid, Ke tone Body, a nd Tria cylglyce rol Me ta bolis m 181 P hos pholipid, Glycos phingolipid, a nd Eicos a noid Me ta bolis m 201 Chole s te rol, Lipoprote in, a nd S te roid Me ta bolis m 219

UNIT IV: Nitrogen Metabolism Chapte r 19: Chapte r 20: Chapte r 21: Chapte r 22:

Amino Acids : Dis pos a l of Nitroge n 245 Amino Acid De gra da tion a nd S ynthe s is 261 Conve rs ion of Amino Acids to S pe cia lize d P roducts Nucle otide Me ta bolis m 291

277

UNIT V: Integration of Metabolism Chapte r 23: Chapte r 24: Chapte r 25: Chapte r 26: Chapte r 27: Chapte r 28:

Me ta bolic Effe cts of Ins ulin a nd Gluca gon The Fe e d–Fa s t Cycle 321 Dia be te s Me llitus 337 Obe s ity 349 Nutrition 357 Vita mins 373

307

UNIT VI: Storage and Expression of Genetic Information Chapte r 29: Chapte r 30: Chapte r 31: Chapte r 32: Chapte r 33:

DNA S tructure , Re plica tion, a nd Re pa ir 395 RNA S tructure , S ynthe s is , a nd P roce s s ing 417 P rote in S ynthe s is 431 Re gula tion of Ge ne Expre s s ion 449 Biote chnology a nd Huma n Dis e a s e 465

Appe ndix: Clinic al Cas e s Inde x 522

489

Bo nus c hapte r o nline ! Chapte r 34: Blood Clotting (Us e your s cra tch-off code provide d in the front of this book for a cce s s to this a nd othe r fre e online re s ource s on

.)

tahir99-VRG & vip.persianss.ir

tahir99-VRG & vip.persianss.ir

UN IT I: Protein Stru ctu re and Fu nction

1

Amino Ac ids I. OVERVIEW P rote ins a re the mos t a bunda nt a nd functiona lly dive rs e mole cule s in living s ys te ms . Virtua lly e ve ry life proce s s de pe nds on this cla s s of ma cromole cule s . For e xa mple , e nzyme s a nd polype ptide hormone s dire ct a nd re gula te me ta bolis m in the body, whe re a s contra ctile prote ins in mus cle pe rmit move me nt. In bone , the prote in colla ge n forms a fra me work for the de pos ition of ca lcium phos pha te crys ta ls , a cting like the s te e l ca ble s in re inforce d concre te . In the bloods tre a m, prote ins , s uch a s he moglobin a nd pla s ma a lbumin, s huttle mole cule s e s s e ntia l to life , whe re a s immunoglobulins fight infe ctious ba cte ria a nd virus e s . In s hort, prote ins dis pla y a n incre dible dive rs ity of functions , ye t a ll s ha re the common s tructura l fe a ture of be ing line a r polyme rs of a mino a cids . This cha pte r de s cribe s the prope rtie s of a mino a cids . Cha pte r 2 e xplore s how the s e s imple building blocks a re joine d to form prote ins tha t ha ve unique thre e -dime ns iona l s tructure s , ma king the m ca pa ble of pe rforming s pe cific biologic functions .

II. S TRUCTURE Although more tha n 300 diffe re nt a mino a cids ha ve be e n de s cribe d in na ture , only 20 a re commonly found a s cons titue nts of ma mma lia n prote ins . [Note : The s e a re the only a mino a cids tha t a re code d for by DNA, the ge ne tic ma te ria l in the ce ll (s e e p. 395).] Ea ch a mino a cid ha s a ca rboxyl group, a prima ry a mino group (e xce pt for proline , which ha s a s e conda ry a mino group), a nd a dis tinctive s ide cha in (“R group”) bonde d to the a -ca rbon a tom (Figure 1.1A). At phys iologic pH (a pproxima te ly 7.4), the ca rboxyl group is dis s ocia te d, forming the ne ga tive ly cha rge d ca rboxyla te ion (– COO – ), a nd the a mino group is protona te d (– NH3 +). In prote ins , a lmos t a ll of the s e ca rboxyl a nd a mino groups a re combine d through pe ptide linka ge a nd, in ge ne ra l, a re not a va ila ble for che mica l re a ction e xce pt for hydroge n bond forma tion (Figure 1.1B). Thus , it is the na ture of the s ide cha ins tha t ultima te ly dicta te s

A

Fre e amino ac id The s e are c o mmo n to all α-amino ac ids .

C CO COOH OH +H N 3

Cα H R

Amino group

S ide c hain is dis tinc tive fo r e ac h amino ac id.

B

Ca rboxyl group

α-Carbo n is linke d to the c arbo xyl, amino , and R g ro ups .

Amino ac ids c o mbine d thro ug h pe ptide linkag e s

NH-CH-CO-NH-CH-CO R

R

S ide c hains de te rmine pro pe rtie s o f pro te ins .

Fig ure 1.1 S tructura l fe a ture s of a mino a cids (shown in their fully protonated form).

1

2

1. Amino Acids the role a n a mino a cid pla ys in a prote in. It is , the re fore , us e ful to cla s s ify the a mino a cids a ccording to the prope rtie s of the ir s ide cha ins , tha t is , whe the r the y a re nonpola r (ha ve a n e ve n dis tribution of e le ctrons ) or pola r (ha ve a n une ve n dis tribution of e le ctrons , s uch a s a cids a nd ba s e s ) a s s hown in Figure s 1.2 a nd 1.3. A. Amino ac ids with no npo lar s ide c hains Ea ch of the s e a mino a cids ha s a nonpola r s ide cha in tha t doe s not ga in or los e protons or pa rticipa te in hydroge n or ionic bonds (s e e Figure 1.2). The s ide cha ins of the s e a mino a cids ca n be thought of a s “oily” or lipid-like , a prope rty tha t promote s hydrophobic inte ra ctions (s e e Figure 2.10, p. 19). 1. Lo c atio n o f no npo lar amino ac ids in pro te ins : In prote ins found

in a que ous s olutions (a pola r e nvironme nt) the s ide cha ins of the nonpola r a mino a cids te nd to clus te r toge the r in the inte rior of the prote in (Figure 1.4). This phe nome non, known as the hydrophobic NONPOLAR S IDE CHAINS COO H +H

3N

C

+H

H

COO H

COO H

pK1 = 2.3 3N

H

C

H

+H

CH3

Alanine

COO H +H

3N

C

+H

H

3N

C

H

H

C

CH3

CH H3 C CH3

N H

3N

C

H

CH2

Is o le uc ine

H

Phe nylalanine

COO H +H

3N

C

CH2

CH2

C

CH2

CH

S

Trypto phan

+H

CH3

COO H C

COO H

CH2

Le uc ine

3N

H

Valine

COO H

CH2

+H

C

CH H3 C CH3

pK2 = 9.6

Glyc ine

3N

COO H

H +H

2N

H2 C

C

H

CH2

CH2

CH3

Me thio nine

Pro line

Fig ure 1.2 Cla s s ifica tion of the 20 a mino a cids commonly found in prote ins , a ccording to the cha rge a nd pola rity of the ir s ide cha ins a t a cidic pH is s hown he re a nd continue s in Figure 1.3. Ea ch a mino a cid is s hown in its fully protona te d form, with dis s ocia ble hydroge n ions re pre s e nte d in re d print. The pK va lue s for the α -ca rboxyl a nd α -a mino groups of the nonpola r a mino a cids a re s imila r to thos e s hown for glycine .

II. S tructure of the Amino Acids

3

UNCHARGED POLAR S IDE CHAINS pK1 = 2.2

COO H +H

COO H +H

C

H

H

C

OH

3N

C

H

H

C

OH

H

3N

C

pK3 = 10.1

OH Tyro s ine

COO H +H

H

3N

C

H

CH2

CH2

C

CH2

O

pK2 = 9.1

Thre o nine

COO H

H

CH2

CH3

S e rine +H

C

COO H +H

3N

3N

NH2

COO H +H

As parag ine

C

H

CH2 pK3 = 10.8

C O

3N

pK1 = 1.7

SH

pK2 = 8.3

NH2

Glutamine

Cys te ine

ACIDIC S IDE CHAINS pK1 = 2.1 COO H +H

pK3 = 9.8

3N

O

C

COO H pK3 = 9.7

H

+H

3N

C

H

CH2

CH2

C

CH2 OH

pK2 = 3.9

C O

As partic ac id

OH

pK2 = 4.3

Glutamatic ac id

BAS IC S IDE CHAINS pK1 = 2.2

pK1 = 1.8 pK3 = 9.2

pK2 = 9.2 COO H

+H

3N

C

pK2 = 9.0

COO H +H

H

CH2

3N

C

+H

H

3N

COO H C

CH2

CH2

C

CH

CH2

CH2

+H N

NH

CH2

CH2

CH2

N

HN

C H

pK2 = 6.0

NH 3 +

pK3 = 10.5

H

H

C NH 2 +

pK3 = 12.5

NH2 His tidine

Lys ine

Arg inine

Fig ure 1.3 Cla s s ifica tion of the 20 a mino a cids commonly found in prote ins , a ccording to the cha rge a nd pola rity of the ir s ide cha ins a t a cidic pH (continue d from Figure 1.2).

4

1. Amino Acids

No npo lar amino ac ids ( ) c lus te r in the inte rio r of s oluble prote ins .

No npo lar amino ac ids ( ) c lus te r o n the s urfac e o f me mbrane pro te ins .

Ce ll me mbrane

Po lar amino ac ids ( ) c lus te r on the s urfac e of s o luble prote ins . S o luble pro te in

Me mbrane pro te in

Fig ure 1.4 Loca tion of nonpola r a mino a cids in s oluble a nd me mbra ne prote ins .

S e c o ndary amino g ro up

Primary amino g ro up

COOH +H N 2

H2 C

C

COOH

H

+H N 3

CH2

C

H

CH3

CH2

Alanine

Pro line

Fig ure 1.5 Compa ris on of the s e conda ry a mino group found in proline with the prima ry a mino group found in othe r a mino a cids s uch a s a la nine . COOH +H N C H 3 CH2

e ffe ct, is the re s ult of the hydrophobicity of the nonpola r R groups , which a ct much like drople ts of oil tha t coa le s ce in a n a que ous e nvironme nt. The nonpola r R groups , thus , fill up the inte rior of the folde d prote in a nd he lp give it its thre e -dime ns iona l s ha pe . Howe ve r, for prote ins tha t a re loca te d in a hydrophobic e nvironme nt, s uch a s a me mbra ne , the nonpola r R groups a re found on the outs ide s urfa ce of the prote in, inte ra cting with the lipid e nvironme nt s e e Figure 1.4. The importa nce of the s e hydrophobic inte ra ctions in s ta bilizing prote in s tructure is dis cus s e d on p. 19.

S ickle ce ll a ne mia , a s ickling dis e a s e of re d blood ce lls , re s ults from the re pla ce me nt of pola r gluta ma te with nonpola r va line a t the s ixth pos ition in the b s ubunit of he moglobin (s e e p. 36).

2. Pro line : P roline diffe rs from othe r a mino a cids in tha t its s ide

cha in a nd a -a mino N form a rigid, five -me mbe re d ring s tructure (Figure 1.5). P roline , the n, ha s a s e conda ry (ra the r tha n a prima ry) a mino group. It is fre que ntly re fe rre d to a s a n “imino a cid.” The unique ge ome try of proline contribute s to the forma tion of the fibrous s tructure of colla ge n (s e e p. 45) a nd ofte n inte rrupts the a -he lice s found in globula r prote ins (s e e p. 26). B. Amino ac ids with unc harg e d po lar s ide c hains The s e a mino a cids ha ve ze ro ne t cha rge a t phys iologic pH, a lthough the s ide cha ins of cys te ine a nd tyros ine ca n los e a proton a t a n a lka line pH (s e e Figure 1.3). S e rine , thre onine , a nd tyros ine e a ch conta in a pola r hydroxyl group tha t ca n pa rticipa te in hydroge n bond forma tion (Figure 1.6). The s ide cha ins of a s pa ra gine a nd gluta mine e a ch conta in a ca rbonyl group a nd a n a mide group, both of which ca n a ls o pa rticipa te in hydroge n bonds . 1. Dis ulfide bo nd: The s ide cha in of cys te ine conta ins a s ulfhydryl

(thiol) group (–SH), which is a n importa nt compone nt of the a ctive s ite of ma ny e nzyme s . In prote ins , the –SH groups of two cys te ine s ca n be oxidize d to form a cova le nt cros s -link ca lled a dis ulfide bond (–S –S–). Two dis ulfide -linke d cys te ine s a re re fe rre d to a s “cys tine .” (Se e p. 19 for a furthe r dis cus s ion of dis ulfide bond forma tion.)

Tyro s ine O H Carbo nyl O g ro up C

Hydro g e n bo nd

Ma n y e xtra ce llula r p rote ins a re s ta b ilize d by dis ulfide bonds . Albumin, a blood prote in tha t functions a s a tra ns porte r for a va rie ty of mole cule s , is a n e xa mple .

2. S ide c hains as s ite s o f attac hme nt fo r o the r c o mpo unds : The

Fig ure 1.6 Hydroge n bond be twe e n the phe nolic hydroxyl group of tyros ine a nd a nothe r mole cule conta ining a ca rbonyl group.

pola r hydroxyl group of s e rine ; thre onine ; a nd, ra re ly, tyros ine , ca n s e rve a s a s ite of a tta chme nt for s tructure s s uch as a phos pha te group. In a ddition, the a mide group of a s pa ra gine , a s we ll a s the hydroxyl group of s e rine or thre onine , ca n s e rve a s a s ite of a tta chme nt for oligos a ccha ride cha ins in glycoprote ins (s e e p. 165).

II. S tructure of the Amino Acids C. Amino ac ids with ac idic s ide c hains The a mino a cids a s pa rtic a nd gluta mic a cid a re proton donors . At physiologic pH, the side chains of these a mino acids a re fully ionized, conta ining a ne ga tive ly cha rge d ca rboxyla te group (–COO – ). The y are , therefore, called aspa rtate or glutama te to emphasize that these amino acids are ne gatively charged at physiologic pH (se e Figure 1.3). D. Amino ac ids with bas ic s ide c hains The s ide cha ins of the ba s ic a mino a cids a cce pt protons (s e e Figure 1.3). At phys iologic pH, the R groups of lys ine a nd a rginine a re fully ionize d a nd pos itive ly cha rge d. In contra s t, his tidine is we a kly ba s ic, a nd the fre e a mino a cid is la rge ly uncha rge d a t phys iologic pH. Howe ve r, whe n his tidine is incorpora te d into a prote in, its R group ca n be e ithe r pos itive ly cha rge d (protona te d) or neutra l, de pe nding on the ionic e nvironme nt provide d by the prote in. This is a n importa nt prope rty of his tidine tha t contribute s to the buffe ring role it pla ys in the functioning of prote ins s uch a s he moglobin (se e p. 31). [Note : His tidine is the only a mino a cid with a s ide cha in tha t ca n ionize within the phys iologic pH ra nge .] E. Abbre viatio ns and s ymbo ls fo r c o mmo nly o c c urring amino ac ids Ea ch a mino a cid na me ha s a n a s s ocia te d thre e -le tte r a bbre via tion a nd a one -le tte r s ymbol (Figure 1.7). The one -le tte r code s a re de te rmine d by the following rule s . 1. Unique firs t le tte r: If only one a mino a cid be gins with a give n le tte r,

the n tha t le tte r is use d a s its s ymbol. For e xa mple , V = va line . 2. Mo s t c o mmo nly o c c urring amino ac ids have prio rity: If more

tha n one a mino a cid be gins with a pa rticula r le tte r, the mos t common of the s e a mino a cids re ce ive s this le tte r a s its s ymbol. For e xa mple , glycine is more common tha n gluta ma te , s o G = glycine . 3. S imilar s o unding name s : S ome one -le tte r s ymbols s ound like the

a mino a cid the y re pre s e nt. For e xa mple , F = phe nyla la nine , or W = tryptopha n (“twyptopha n” a s Elme r Fudd would s a y). 4. Le tte r c lo s e to initial le tte r: For the re ma ining a mino a cids , a one -

le tte r s ymbol is a s s igne d tha t is a s clos e in the a lpha be t a s pos s ible to the initia l le tte r of the a mino a cid, for e xa mple , K = lys ine . Furthe rmore , B is a s s igne d to As x, s ignifying e ithe r a s pa rtic a cid or a s pa ra gine , Z is a s s igne d to Glx, s ignifying e ithe r gluta mic a cid or gluta mine , a nd X is a s s igne d to a n unide ntifie d a mino a cid.

5

1

Unique firs t letter:

C ys te ine H is tid ine

= = = = = =

Is ole uc ine Me thionine S e rine V aline

2

= = = = =

= = = = = = = =

S V

Ala Gly Le u Pro Thr

A

= = = = =

G L P T

Arg As n As p Glu Gln Phe Tyr Trp

= = = = = = = =

R N D E Q

F Y W

(“aR g inine ”) (c o ntains N) ("as parD ic ") ("g lutE mate ") (“Q -tamine ”) (“F enylalanine”) (“tY ro s ine ”) (double ring in the mo le c ule )

Letter clos e to initial letter:

As partate o r as parag ine Glutamate o r g lutamine Lys ine Unde te rmine d amino ac id

=

As x =

B (ne ar A)

=

Glx =

Z

= =

Lys =

K (ne ar L) X

Fig ure 1.7 Abbre via tions a nd s ymbols for the commonly occurring a mino a cids .

O CO C H

F. Optic al pro pe rtie s o f amino ac ids The a -ca rbon of a n a mino a cid is a tta che d to four diffe re nt che mica l groups (a s ymme tric) a nd is , the re fore , a chira l, or optica lly a ctive ca rbon a tom. Glycine is the e xce ption be ca us e its a -ca rbon ha s two hydroge n s ubs titue nts . Amino a cids with a n a s ymme tric ce nte r a t the a -ca rbon ca n e xis t in two forms , de s igna te d D a nd L, tha t a re mirror ima ge s of e a ch othe r (Figure 1.8). The two forms in e a ch pa ir a re te rme d s te re ois ome rs , optica l is ome rs , or e na ntiome rs . All a mino a cids found in prote ins a re of the l configura tion. Howe ve r, D-a mino a cids a re found in s ome a ntibiotics a nd in ba cte ria l ce ll wa lls . (S e e p. 252 for a dis cus s ion of D-a mino a cids .)

M

Similar s ounding names :

Ar g inine As p a ra g in e As pa rta te Gluta ma te Gluta mine Phenylalanine Tyros ine Tryptop ha n

4

C H I

= = = = = =

Mo s t c o mmo nly o c c urring amino ac ids have prio rity:

Alanine G lyc ine Le uc ine P ro line T hre onine

3

Cys His Ile Me t Ser Val

+H3 N

l

L-A

H

C H3 ne ani

HO

OC H C

H C 3 D- A la

Fig ure 1.8 D a nd L forms of a la nine a re mirror ima ge s .

NH

3

n in

e

+

6

1. Amino Acids

III. ACIDIC AND BAS IC PROPERTIES OF AMINO ACIDS Amino a cids in a que ous s olution conta in we a kly a cidic a -ca rboxyl groups a nd we a kly ba s ic a -a mino groups . In a ddition, e a ch of the a cidic a nd ba s ic a mino a cids conta ins a n ioniza ble group in its s ide cha in. Thus , both fre e a mino a cids a nd s ome a mino a cids combine d in pe ptide linka ge s ca n a ct a s buffe rs . Re ca ll tha t a cids ma y be de fine d a s proton donors a nd ba s e s a s proton a cce ptors . Acids (or ba s e s ) de s cribe d a s “we a k” ionize to only a limite d e xte nt. The conce ntra tion of protons in a que ous s olution is e xpre s s e d a s pH, whe re pH = log 1/[H+] or – log [H+]. The qua ntita tive re la tions hip be twe e n the pH of the s olution a nd conce ntra tion of a we a k a cid (HA) a nd its conjuga te ba s e (A– ) is de s cribe d by the He nde rs on-Ha s s e lba lch e qua tion. OH–

A. De rivatio n o f the e quatio n

H 2O

FORM I (ac e tic ac id, HA)

FORM II

H + (ac e tate , A– )

Buffe r re gion

[II] > [I]

1.0

HA we a k a cid

← →

H+ proton

[I] = [II]

Ka

pKa = 4.8

0.5

[I] > [II] 0 0

3

4

5

6

pH

Fig ure 1.9 Titra tion curve of a ce tic a cid.

7

A– s a lt form or conjuga te ba s e

+

The “s a lt” or conjuga te ba s e , A– , is the ionize d form of a we a k a cid. By de finition, the dis s ocia tion cons ta nt of the a cid, Ka , is



Equivale nts OH adde d

Cons ide r the re le a s e of a proton by a we a k a cid re pre s e nte d by HA:

CH3 COO –

CH3 COO H

[H+] [A– ] [HA]

[Note : The la rge r the Ka , the s tronge r the a cid, be ca us e mos t of the HA ha s dis s ocia te d into H+ a nd A– . Conve rs e ly, the s ma lle r the Ka , the le s s a cid ha s dis s ocia te d a nd, the re fore , the we a ke r the a cid.] By s olving for the [H+] in the a bove e qua tion, ta king the loga rithm of both s ide s of the e qua tion, multiplying both s ide s of the e qua tion by –1, a nd s ubs tituting pH = – log [H+ ] a nd pKa = – log Ka , we obta in the He nde rs on-Ha s s e lba lch e qua tion:

pH

[A– ] pKa + log [HA]

B. Buffe rs A buffe r is a s olution tha t re s is ts cha nge in pH following the a ddition of a n a cid or ba s e . A buffe r ca n be cre a te d by mixing a we a k a cid (HA) with its conjuga te ba s e (A– ). If a n a cid s uch a s HCl is a dde d to a buffe r, A– ca n ne utra lize it, be ing conve rte d to HA in the proce s s. If a ba s e is a dde d, HA ca n ne utra lize it, be ing conve rte d to A– in the proce s s . Ma ximum buffe ring ca pa city occurs a t a pH e qua l to the pKa , but a conjuga te a cid–ba s e pa ir ca n s till s e rve a s a n e ffe ctive buffe r whe n the pH of a s olution is within a pproxima te ly ±1 pH unit of the pKa . If the a mounts of HA a nd A– a re e qua l, the pH is e qua l to the pKa . As s hown in Figure

III. Acidic a nd Ba s ic P rope rtie s of Amino Acids



OH

7



H 2O

COO H +H N C H 3

OH COO +H N C H 3

CH3

H+

FORM I



CH3 FORM II

pK1 = 2.3

H 2O COO H2 N C H

H+



CH3 FORM III

pK2 = 9.1

Alanine in ac id s o lutio n (pH le s s than 2)

Alanine in ne utral s o lutio n (pH appro ximate ly 6)

Alanine in bas ic s o lutio n (pH g re ate r than 10)

Ne t c harg e = +1

Ne t c harg e = 0 (is o e le c tric fo rm)

Ne t c harg e = – 1

Fig ure 1.10 Ionic forms of a la nine in a cidic, ne utra l, a nd ba s ic s olutions . 1.9, a s olution conta ining a ce tic a cid (HA = CH3 – COOH) a nd a ce ta te (A– = CH3 – COO – ) with a pKa of 4.8 re s is ts a cha nge in pH from pH 3.8 to 5.8, with ma ximum buffe ring a t pH 4.8. At pH va lue s le s s tha n the pKa , the protona te d a cid form (CH3 – COOH) is the pre domina nt s pe cie s in s olution. At pH va lue s gre a te r tha n the pKa , the de protona te d ba s e form (CH3 – COO – ) is the pre domina nt s pe cie s . C. Titratio n o f an amino ac id 1. Dis s o c iatio n o f the c arbo xyl g ro up: The titra tion curve of a n a mino a cid ca n be a na lyze d in the s a me wa y a s de s cribe d for a ce tic a cid. Cons ide r a la nine , for e xa mple , which conta ins a n ioniza ble a -ca rboxyl a nd a -a mino group. [Note : Its –CH3 R group is nonioniza ble .] At a low (a cidic) pH, both of the s e groups a re protona te d (s hown in Figure 1.10). As the pH of the s olution is ra is e d, the – COOH group of form I ca n dis s ocia te by dona ting a proton to the me dium. The re le a s e of a proton re s ults in the forma tion of the ca rboxyla te group, – COO – . This s tructure is s hown a s form II, which is the dipola r form of the mole cule (s e e Figure 1.10). This form, a ls o ca lle d a zwitte rion, is the is oe le ctric form of a la nine , tha t is , it ha s a n ove ra ll (ne t) cha rge of ze ro. 2. Applic atio n o f the He nde rs o n-Has s e lbalc h e quatio n: The dis s oci-

a tion cons ta nt of the ca rboxyl group of a n a mino a cid is ca lle d K1 , ra the r tha n Ka , be ca us e the mole cule conta ins a s e cond titra ta ble group. The He nde rs on-Ha s s e lba lch e qua tion ca n be us e d to a na lyze the dis s ocia tion of the ca rboxyl group of a la nine in the s a me wa y a s de s cribe d for a ce tic a cid:

K1

[H+] [II] [I]

whe re I is the fully protona te d form of a la nine , a nd II is the is oe le ctric form of a la nine (s e e Figure 1.10). This e qua tion ca n be re a rra nge d a nd conve rte d to its loga rithmic form to yie ld:

8

1. Amino Acids

pH

pK1 + log

[II] [I]

3. Dis s o c iatio n o f the amino g ro up: The s e cond titra ta ble group of

COO H2 N C H

a la nine is the a mino (– NH3 +) group s hown in Figure 1.10. This is a much we a ke r a cid tha n the – COOH group a nd, the re fore , ha s a much s ma lle r dis s ocia tion cons ta nt, K2 . [Note : Its pKa is , the re fore , la rge r.] Re le a s e of a proton from the protona te d a mino group of form II re s ults in the fully de protona te d form of a la nine , form III (s e e Figure 1.10).



CH3

4. pKs o f alanine : The s e que ntia l dis s ocia tion of protons from the ca r-

FORM III Re gion of buffe ring

Re gion of buffe ring [II] = [III]

Equivale nts OH– adde d

2.0

pI = 5.7

1.5 [I] = [II]

1.0

pK p K2 = 9. 9.1 pK1 = 2.3

0.5 0

0

2

4

6

8

10

pH p

COO H +H N C H 3

COO +H N C H 3



CH3

CH3

FORM I

FORM II

boxyl a nd a mino groups of a la nine is s umma rize d in Figure 1.10. Ea ch titra ta ble group ha s a pKa tha t is nume rica lly e qua l to the pH a t which e xa ctly one ha lf of the protons ha ve be e n re move d from tha t group. The pKa for the mos t a cidic group (–COOH) is pK1 , whe re a s the pKa for the ne xt mos t a cidic group (– NH3 +) is pK2 . [Note : The pKa of the a -ca rboxyl group of a mino a cids is a pproxima te ly 2, whe rea s tha t of the a -a mino is a pproxima te ly 9.] 5. Titratio n c urve of alanine : By a pplying the He nde rs on-Ha s s e lba lch

e qua tion to e a ch dis s ocia ble a cidic group, it is pos s ible to ca lcula te the comple te titra tion curve of a we a k a cid. Figure 1.11 s hows the cha nge in pH tha t occurs during the a ddition of ba se to the fully protona te d form of a la nine (I) to produce the comple te ly de protona te d form (III). Note the following: a. Buffe r pairs : The – COOH/– COO – pa ir ca n s e rve a s a buffe r

in the pH re gion a round pK1 , a nd the – NH3 +/– NH2 pa ir ca n buffe r in the re gion a round pK2 . b. Whe n pH = pK: Whe n the pH is e qua l to pK1 (2.3), e qua l

Fig ure 1.11 The titra tion curve of a la nine .

a mounts of forms I a nd II of a la nine e xis t in s olution. Whe n the pH is e qua l to pK2 (9.1), e qua l a mounts of forms II a nd III a re pre s e nt in s olution. c . Is o e le c tric po int: At ne utra l pH, a la nine e xis ts pre domina ntly

a s the dipola r form II in which the a mino a nd ca rboxyl groups a re ionize d, but the ne t cha rge is ze ro. The is oe le ctric point (pI) is the pH a t which a n a mino a cid is e le ctrica lly ne utra l, tha t is , in which the s um of the pos itive cha rge s e qua ls the s um of the ne ga tive cha rge s . For a n a mino a cid, s uch a s a la nine , tha t ha s only two dis s ocia ble hydroge ns (one from the a -ca rboxyl a nd one from the a -a mino group), the pI is the a ve ra ge of pK1 a nd pK2 (pI = [2.3 + 9.1]/2 = 5.7) a s s hown in Figure 1.11. The pI is , thus , midwa y be twe e n pK1 (2.3) a nd pK2 (9.1). pI corre s ponds to the pH a t which the form II (with a ne t cha rge of ze ro) pre domina te s a nd a t which the re a re a ls o e qual a mounts of forms I (ne t cha rge of +1) a nd III (ne t cha rge of –1).

III. Acidic a nd Ba s ic P rope rtie s of Amino Acids

Se pa ra tion of pla s ma prote ins by cha rge typica lly is done a t a pH a bove the pI of the ma jor prote ins . Thus , the cha rge on the prote ins is ne ga tive . In a n e le ctric fie ld, the prote ins will move towa rd the pos itive e le ctrode a t a ra te de te rmine d by the ir ne t ne ga tive cha rge . Va ria tions in the mobility pa tte rn a re s ugge s tive of ce rta in dis e a s e s .

9

A

[HCO3 – ] pH = pK + lo g [CO2 ] An inc re as e in HCO3 – c aus e s the pH to ris e . Pulmo nary o bs truc tio n c aus e s an inc re as e in c arbo n dio xide , whic h c aus e s the pH to fall, re s ulting in re s pirato ry ac ido s is .

6. Ne t c harg e o f amino ac ids at ne utral pH: At phys iologic pH,

a mino a cids ha ve a ne ga tive ly cha rge d group (– COO – ) a nd a pos itive ly cha rge d group (– NH3 + ), both a tta che d to the a -ca rbon. [Note : Gluta ma te , a s pa rta te , his tidine , a rginine , a nd lys ine ha ve a dditiona l pote ntia lly cha rge d groups in the ir s ide cha ins .] S ubs ta nce s s uch a s a mino a cids tha t ca n a ct e ithe r a s a n a cid or a ba s e a re de fine d a s a mphote ric a nd a re re fe rre d to a s a mpholyte s (a mphote ric e le ctrolyte s ).

D. Othe r applic atio ns o f the He nde rs o n-Has s e lbalc h e quatio n The He nde rs on-Ha s s e lba lch e qua tion ca n be us e d to ca lcula te how the pH of a phys iologic s olution re s ponds to cha nge s in the conce ntra tion of a we a k a cid a nd/or its corre s ponding “s a lt” form. For e xa mple , in the bica rbona te buffe r s ys te m, the He nde rs onHa s s e lba lch e qua tion pre dicts how s hifts in the bica rbona te ion conce ntra tion, [HCO 3 – ], a nd CO 2 influe nce pH (Figure 1.12A). The e qua tion is a ls o us e ful for ca lcula ting the a bunda nce of ionic forms of a cidic a nd ba s ic drugs . For e xa mple , mos t drugs a re e ithe r we a k a cids or we a k ba s e s (Figure 1.12B). Acidic drugs (HA) re le a s e a proton (H+), ca us ing a cha rge d a nion (A– ) to form. HA

→ ←

BICARBONATE AS A BUFFER

LUNG ALVEOLI

CO2 + H2 O

B

H2 CO3

DRUG ABS ORPTION pH = pK + log

Uncharg ed drug s g enerally c ro s s me mbrane s mo re rapidly than do c harg e d mole c ule s . S TOMACH

H+ + A– Lipid me mbrane

H+

→ ←

– [Drug ] [Drug -H]

At the pH o f the s to mac h (1.5), a drug like as pirin (we ak ac id, pK = 3.5) will be larg e ly protonate d (COOH) and, thus , unc harg e d.

We a k ba s e s (BH+) ca n a ls o re le a s e a H+. Howe ve r, the protona te d form of ba s ic drugs is us ua lly cha rge d, a nd the los s of a proton produce s the uncha rge d ba s e (B). BH+

H+ + HCO3 -

B + H+

H+ HA

A drug pa s s e s through me mbra ne s more re a dily if it is uncha rge d. Thus , for a we a k a cid, s uch a s a s pirin, the uncha rge d HA ca n pe rme a te through me mbra ne s , but A– ca nnot. For a we a k ba s e , s uch a s morphine , the uncha rge d form, B, pe ne tra te s through the ce ll me mbra ne , but BH+ doe s not. The re fore , the e ffe ctive conce ntra tion of the pe rme a ble form of e a ch drug a t its a bs orption s ite is de te rmine d by the re la tive conce ntra tions of the cha rge d (impe rme a nt) a nd uncha rge d (pe rme a nt) forms . The ra tio be twe e n the two forms is de te rmine d by the pH a t the s ite of a bs orption, a nd by the s tre ngth of the we a k a cid or ba s e , which is re pre s e nte d by the pKa of the ioniza ble group. The He nde rs on-Ha s s e lba lch e qua tion is us e ful in de te rmining how much drug is found on e ithe r s ide of a me mbra ne tha t s e pa ra te s two compa rtme nts tha t diffe r in pH, for e xa mple , the s toma ch (pH 1.0–1.5) a nd blood pla s ma (pH 7.4).

A-

H+

AH+

HA

LUMEN OF S TOMACH

BLOOD

Fig ure 1.12 The He nde rs on-Ha s s e lba lch e qua tion is us e d to pre dict: A, cha nge s in pH a s the conce ntra tions of HCO 3 – or CO 2 a re a lte re d, or B, the ionic forms of drugs .

10

1. Amino Acids

A Linke d c o nc e pt bo xe s Amino ac ids (fully protona te d)

ca n Re le as e H+

nc e pts c ro s s -linke d B Co within a map De g radatio n o f bo dy pro te in

is p rod u c e d by

S imultane o us s ynthe s is and de g radatio n

Amino ac id po o l

Pro te in turno ve r

le a d s to

IV. CONCEPT MAPS S tude nts s ome time s vie w bioche mis try a s a lis t of fa cts or e qua tions to be me morize d, ra the r tha n a body of conce pts to be unde rs tood. De ta ils provide d to e nrich unde rs ta nding of the s e conce pts ina dve rte ntly turn into dis tra ctions . Wha t s e e ms to be mis s ing is a roa d ma p—a guide tha t provide s the s tude nt with a n unde rs ta nding of how va rious topics fit toge the r to ma ke s e ns e . The re fore , a s e rie s of bioche mica l conce pt ma ps ha ve be e n cre a te d to gra phica lly illus tra te rela tions hips be twe e n ide a s pre s e nte d in a cha pte r a nd to s how how the informa tion ca n be groupe d or orga nize d. A conce pt ma p is , thus , a tool for vis ua lizing the conne ctions be twe e n conce pts . Ma te ria l is re pre s e nte d in a hie ra rchic fa s hion, with the mos t inclus ive , mos t ge ne ra l concepts a t the top of the ma p a nd the more s pe cific, le s s ge ne ra l conce pts a rra nge d be ne a th. The conce pt ma ps ide a lly function a s te mpla te s or guide s for orga nizing informa tion, s o the s tude nt ca n re a dily find the bes t wa ys to inte gra te ne w informa tion into knowle dge the y a lre a dy pos s e s s . A. Ho w is a c o nc e pt map c o ns truc te d? 1. Co nc e pt bo xe s and links : Educa tors de fine conce pts a s “pe r-

S ynthe s is o f bo dy pro te in

is c o ns um e d by

Amino ac id po o l

C Co nc e pts c ro s s -linke d to o the r c hapte rs in the bo o k

. . . ho w the pro te in fo lds into its native c o nfo rmatio n

S truc ture o f Pro te ins

2

ce ive d re gula ritie s in e ve nts or obje cts .” In the bioche mica l ma ps , conce pts include a bs tra ctions (for e xa mple , fre e e ne rgy), proce s s e s (for e xa mple , oxida tive phos phoryla tion), a nd compounds (for e xa mple , glucos e 6-phos pha te ). The s e broa dly de fine d conce pts a re prioritize d with the ce ntra l ide a pos itione d a t the top of the pa ge . The conce pts tha t follow from this ce ntra l ide a a re the n dra wn in boxe s (Figure 1.13A). The s ize of the type indica te s the re la tive importa nce of e a ch ide a . Line s a re dra wn be twe e n conce pt boxe s to s how which a re re la te d. The la be l on the line de fine s the re la tions hip be twe e n two conce pts , s o tha t it re a ds a s a va lid s ta te me nt, tha t is , the conne ction cre a te s me a ning. The line s with a rrowhe a ds indica te in which dire ction the conne ction s hould be re a d (Figure 1.14). 2. Cro s s -links : Unlike line a r flow cha rts or outline s , conce pt ma ps

ma y conta in cros s -links tha t a llow the re a de r to vis ua lize comple x re la tions hips be twe e n ide a s re pre s e nte d in diffe re nt pa rts of the ma p (Figure 1.13B), or be twe e n the ma p a nd othe r cha pte rs in this book (Figure 1.13C). Cros s -links ca n, thus , ide ntify conce pts tha t a re ce ntra l to more tha n one topic in bioche mis try, e mpowe ring s tude nts to be e ffe ctive in clinica l s itua tions a nd on the Unite d S ta te s Me dica l Lice ns ure Exa mina tion (US MLE) or othe r e xa mina tions tha t re quire inte gra tion of ma te ria l. S tude nts le a rn to vis ua lly pe rce ive nonline a r re la tions hips be twe e n fa cts , in contra s t to cros s -re fe re ncing within line a r te xt.

V. CHAPTER S UMMARY Fig ure 1.13 S ymbols us e d in conce pt ma ps .

Ea ch a mino a cid ha s a n a -c arbo xyl g ro up a nd a prima ry a -amino g ro u p (e xce pt for proline , which ha s a s e c o ndary amino g ro up ). At phys iologic pH, the a -ca rboxyl group is dis s ocia te d, forming the ne ga tive ly cha rge d ca rboxyla te ion (– COO – ), a nd the a -a mino group is protona te d (– NH3 +). Ea ch a mino a cid a ls o conta ins one of 20 dis tinctive

V. Cha pte r S umma ry

11

Amino ac ids a re compos e d of

α-C a r b o x y l g r o u p (–C O O H )

α-Am in o g r o u p (–N H 2 )

is

whe n protona te d ca n

+

S id e c h a in s (R g r o u p s ) (2 0 d iffe r e n t o n e s )

Re le as e H and act as

is

De pro to nate d (COO– ) at phys io lo g ic pH

Pro to nate d (NH3+ ) at phys io lo g ic pH

We ak ac ids

groupe d a s

described by He nde rs o n-Has s e lbalc h e quatio n: [A– ] pH = pKa + lo g [HA] No npo lar s ide c hains

Unc harg e d po lar s ide c hains

Ac idic s ide c hains

Bas ic s ide c hains

Ala nine Glycine Is ole ucine Le ucine Me thionine Phe nyla la nine Proline Tryptopha n Va line

As pa ra gine Cys te ine Gluta mine S e rine Thre onine Tyros ine

As pa rtic a cid Gluta mic a cid

Arginine His tidine Lys ine

cha ra cte rize d by S ide c hain dis s o c iate s to –COO– at phys io lo g ic pH

predicts

Buffe ring c apac ity predicts

cha ra cte rize d by S ide c hain is pro to nate d and g e ne rally has a po s itive c harg e at phys io lo g ic pH

Buffe ring o c c urs ±1 pH unit o f pKa predicts

found

found

found

found Maximal buffe r whe n pH = pKa

On the o uts ide o f pro te ins that func tio n in an aque o us e nviro nme nt and in the inte rio r o f me mbrane -as s o c iate d pro te ins

predicts In the inte rio r o f pro te ins that func tio n in an aque o us e nviro nme nt and o n the s urfac e o f pro te ins (s uc h as me mbrane pro te ins ) that inte rac t with lipids

In pro te ins , mo s t α-COO– and α-NH3+ o f amino ac ids are c o mbine d thro ug h pe ptide bo nds .

The re fo re , the s e g ro ups are no t available fo r c he mic al re ac tio n.

Fig ure 1.14 Ke y conce pt ma p for a mino a cids .

pH = pKa whe n [HA] = [A– ]

Thus , the c he mic al nature o f the s ide c hain de te rmine s the ro le that the amino ac id plays in a pro te in, partic ularly . . .

S truc ture o f Pro te ins . . . ho w the pro te in fo lds into its native c o nfo rmatio n.

2

12

1. Amino Acids

s ide c hains a tta che d to the a -ca rbon a tom. The che mica l na ture of this R group de te rmine s the function of a n a mino a cid in a prote in a nd provide s the ba s is for cla s s ifica tion of the a mino a cids a s no npo lar, unc harg e d po lar, ac idic (po lar ne g ative ), or bas ic (po lar po s itive ). All fre e a mino a cids , plus cha rge d a mino a cids in pe p-

tide cha ins , ca n s e rve a s buffe rs . The qua ntita tive re la tions hip be twe e n the pH of a s olution a nd the conce ntra tion of a we a k a cid (HA) a nd its conjuga te ba s e (A– ) is de s cribe d by the He nde rs o n-Has s e lbalc h e quatio n . Buffe ring occurs within ±1 pH unit of the pKa a nd is ma xima l whe n pH = pKa , a t which [A– ] = [HA]. The a -ca rbon of e a ch a mino a cid (e xce pt glycine ) is a tta che d to four diffe re nt che mica l groups a nd is , the re fore , a c hiral, or o ptic ally ac tive ca rbon a tom. The L-form of a mino a cids is found in prote ins s ynthe s ize d by the huma n body.

S tudy Que s tio ns Choos e the ONE be s t a ns we r.

Equivale nts OH– adde d

1.1 Which one of the following s ta te me nts conce rning the titra tion curve for a nonpola r a mino a cid is corre ct? The le tte rs A through D de s igna te ce rta in re gions on the curve be low. 2.0 D

1.5 1.0

C

0.5

B A

0

0

2

4

6

8

Corre ct a ns we r = C. C re pre s e nts the is oe le ctric point, or pI, a nd a s s uch is midwa y be twe e n pK1 a nd pK2 for a nonpola r a mino a cid. The a mino a cid is fully protona te d a t P oint A. P oint B re pre s e nts a re gion of ma ximum buffe ring, a s doe s P oint D. Lys ine is a ba s ic a mino a cid, a nd ha s a n ioniza ble s ide cha in.

10

pH

A. P oint A re pre s e nts the re gion whe re the a mino a cid is de protona te d. B. P oint B re pre s e nts a re gion of minima l buffe ring. C. P oint C re pre s e nts the re gion whe re the ne t cha rge on the a mino a cid is ze ro. D. P oint D re pre s e nts the pK of the a mino a cid’s ca rboxyl group. E. The a mino a cid could be lys ine . 1.2 Which one of the following s ta te me nts conce rning the pe ptide s hown be low is corre ct? Va l-Cys -Glu-S e r-As p-Arg-Cys A. The pe ptide conta ins a s pa ra gine . B. The pe ptide conta ins a s ide cha in with a s e conda ry a mino group. C. The pe ptide conta ins a s ide cha in tha t ca n be phos phoryla te d. D. The pe ptide ca nnot form a n inte rna l dis ulfide bond. E. The pe ptide would move to the ca thode (ne ga tive e le ctrode ) during e le ctrophore s is a t pH 5. 1.3 A 2-ye a r-old child pre s e nts with me ta bolic a cidos is a fte r inge s ting a n unknown numbe r of fla vore d a s pirin ta ble ts . At pre s e nta tion, he r blood pH wa s 7.0. Give n tha t the pKa of a s pirin (s a licylic a cid) is 3, ca lcula te the ra tio of its ionize d to un-ionize d forms a t pH 7.0.

Corre ct a ns we r = C. The hydroxyl group of s e rine ca n a cce pt a phos pha te group. As p is a s pa rta te . P roline conta ins a s e conda ry a mino group. The two cys te ine re s idue s ca n, unde r oxidizing conditions , form a dis ulfide (cova le nt) bond. The ne t cha rge on the pe ptide a t pH 5 is ne ga tive , a nd it would move to the a node .

Corre ct a ns we r = 10,000 to 1. pH = pKa + log [A–]/[HA]. The re fore , 7 = 3 + × a nd × = 4. The ra tio of A– (ionize d) to HA (unionize d), the n, is 10,000 to 1 be ca us e the log of 10,000 is 4.

S truc ture o f Pro te ins I. OVERVIEW The 20 a mino a cids commonly found in prote ins a re joine d toge the r by pe ptide bonds . The line a r s e que nce of the linke d a mino a cids conta ins the informa tion ne ce s s a ry to ge ne ra te a prote in mole cule with a unique thre e -dime ns iona l s ha pe . The comple xity of prote in s tructure is be s t a na lyze d by cons ide ring the mole cule in te rms of four orga niza tiona l le ve ls : prima ry, s e conda ry, te rtia ry, a nd qua te rna ry (Figure 2.1). An e xa mina tion of the s e hie ra rchie s of incre a s ing comple xity ha s re ve a le d tha t ce rta in s tructura l e le me nts a re re pe a te d in a wide va rie ty of prote ins , s ugge s ting tha t the re a re ge ne ra l “rule s ” re ga rding the wa ys in which prote ins a chie ve the ir na tive , functiona l form. The s e re pe a te d s tructura l e le me nts ra nge from s imple combina tions of α -he lice s a nd β-s he e ts forming s ma ll motifs , to the comple x folding of polype ptide doma ins of multifunctiona l prote ins (s e e p. 19).

II. PRIMARY S TRUCTURE OF PROTEINS

2 H H

H

N C

C N C

H

H

O

Primary s truc ture

1

C

CH3

N C O H C O N C CH O

N H

C

C

N R C H N H O C R C

C R

e c o ndary 2 Ss truc ture

O

O

C C

NC H C R N H

3

Te rtiary s truc ture

4

Quate rnary s truc ture

The s e que nce of a mino a cids in a prote in is ca lle d the prima ry s tructure of the prote in. Unde rs ta nding the prima ry s tructure of prote ins is importa nt be ca us e ma ny ge ne tic dis e a s e s re s ult in prote ins with a bnorma l a mino a cid s e que nce s , which ca us e imprope r folding a nd los s or impa irme nt of norma l function. If the prima ry s tructure s of the norma l a nd the muta te d prote ins a re known, this informa tion ma y be us e d to dia gnos e or s tudy the dis e a s e . A. Pe ptide bo nd In prote ins , a mino a cids a re joine d cova le ntly by pe ptide bonds , which a re a mide linka ge s be twe e n the α -ca rboxyl group of one a mino a cid a nd the α -a mino group of a nothe r. For e xa mple , va line a nd a la nine ca n form the dipe ptide va lyla la nine through the forma tion of a pe ptide bond (Figure 2.2). P e ptide bonds a re re s is ta nt to conditions tha t de na ture prote ins , s uch a s he a ting a nd high conce ntra tions of ure a (s e e p. 20). Prolonge d e xpos ure to a s trong a cid or ba s e a t e le va te d te mpe ra ture s is re quire d to bre a k the s e bonds none nzymica lly.

Fig ure 2.1 Four hie ra rchie s of prote in s tructure .

13

14

2. S tructure of P rote ins 1. Naming the peptide: By convention, the free amino end (N-terminal)

rmatio n o f the A Fo pe ptide bo nd CH3 H

H3 C CH +H N 3

COO–

C

+H

3N

C

COO –

H

CH3

Valine

Alanine H2 O

Free amino end of peptide

Free carboxyl end of peptide CH3

2. Charac te ris tic s o f the pe ptide bo nd: The pe ptide bond ha s a pa r-

H3 C CH +

H3 N C

C

H

O

H

H

N

C

COO –

CH3

Valylalanine Peptide bond

te ris tic s o f the B Charac pe ptide bo nd Trans pe ptide bo nd

O

Cα C N

R



H

of the peptide chain is writte n to the le ft and the free carboxyl end (C-te rmina l) to the right. The re fore , a ll a mino a cid se que nce s a re rea d from the N- to the C-terminal end of the pe ptide. For example , in Figure 2.2A, the orde r of the a mino a cids is “va line , a la nine .” Linka ge of many a mino acids through peptide bonds re sults in an unbra nche d cha in ca lle d a polype ptide . Ea ch compone nt a mino acid in a polype ptide is ca lle d a “residue” be cause it is the portion of the amino acid rema ining afte r the atoms of water are lost in the formation of the peptide bond. When a polype ptide is name d, all amino acid residues have their suffixes (-ine, -an, -ic, or -ate) change d to -yl, with the e xception of the C-te rmina l amino acid. For example , a tripe ptide compos e d of a n N-te rmina l va line , a glycine , a nd a C-terminal leucine is called valylglycylleucine.

R R

Cis pe ptide bo nd

R

Cα Cα C N O H

Pe ptide bo nds in pro te ins Partial do uble -bo nd c harac te r Rig id and planar Trans c o nfig uratio n Unc harg e d but po lar

Fig ure 2.2 A. Forma tion of a pe ptide bond, s howing the s tructure of the dipe ptide va lyla la nine . B. Cha ra cte ris tics of the pe ptide bond.

tia l double -bond cha ra cte r, tha t is , it is s horte r tha n a s ingle bond a nd is rigid a nd pla na r (Figure 2.2B). This pre ve nts fre e rota tion a round the bond be twe e n the ca rbonyl ca rbon a nd the nitroge n of the pe ptide bond. Howe ve r, the bonds be twe e n the α -ca rbons a nd the α -a mino or α -ca rboxyl groups ca n be fre e ly rota te d (a lthough the y a re limite d by the s ize a nd cha ra cte r of the R groups ). This a llows the polype ptide cha in to a s s ume a va rie ty of pos s ible configura tions . The pe ptide bond is a lmos t a lwa ys a tra ns bond (ins te a d of cis , s e e Figure 2.2B), in la rge pa rt be ca us e of s te ric inte rfe re nce of the R groups whe n in the cis pos ition. 3. Po larity o f the pe ptide bo nd: Like a ll a mide linka ge s , the – C = O

a nd – NH groups of the pe ptide bond a re uncha rge d a nd ne ithe r a cce pt nor re le a s e protons ove r the pH ra nge of 2–12. Thus , the cha rge d groups pre s e nt in polype ptide s cons is t s ole ly of the N-te rmina l (α -a mino) group, the C-te rmina l (α -ca rboxyl) group, a nd a ny ionize d groups pre s e nt in the s ide cha ins of the cons titue nt a mino a cids . The – C = O a nd – NH groups of the pe ptide bond a re pola r, howe ve r, a nd a re involve d in hydroge n bonds (for e xa mple , in α -he lice s a nd β-s he e ts ), a s de s cribe d on pp. 16–17. B. De te rminatio n o f the amino ac id c o mpo s itio n o f a po lype ptide The firs t s te p in de te rmining the prima ry s tructure of a polype ptide is to ide ntify a nd qua ntita te its cons titue nt a mino a cids . A purifie d s a mple of the polype ptide to be a na lyze d is firs t hydrolyze d by s trong a cid a t 110°C for 24 hours . This tre a tme nt cle a ve s the pe ptide bonds a nd re le a s e s the individua l a mino a cids , which ca n be s e pa ra te d by ca tion-e xcha nge chroma togra phy. In this te chnique , a mixture of a mino a cids is a pplie d to a column tha t conta ins a re s in to which a ne ga tive ly cha rge d group is tightly a tta che d. [Note : If the a tta che d group is pos itive ly cha rge d, the column be come s a n a nion-e xcha nge column.] The a mino a cids bind to the column with diffe re nt a ffinitie s , de pe nding on the ir cha rge s , hydrophobicity, a nd othe r cha ra cte ris tics . Ea ch a mino a cid is s e que ntia lly re le a s e d from the chroma togra phy column by e luting with s olutions of incre a s ing ionic s tre ngth a nd pH (Figure 2.3). The s e pa ra te d a mino a cids conta ine d in the e lua te from the column a re qua ntita ted by he a ting the m with ninhydrin (a re a ge nt tha t forms a purple compound with mos t

II. P rima ry S tructure of P rote ins

15

a mino a cids , a mmonia , a nd a mine s ). The a mount of e a ch a mino a cid is de te rmine d s pe ctrophotome trica lly by me a s uring the a mount of light a bs orbe d by the ninhydrin de riva tive . The a na lys is de s cribe d a bove is pe rforme d us ing a n a mino a cid a na lyze r, a n a utoma te d ma chine whos e compone nts a re de picte d in Figure 2.3.

Buffe r pump

S ample inje c tio n

Io n e xc hang e c o lumn

C. S e que nc ing o f the pe ptide fro m its N-te rminal e nd S e que ncing is a s te pwis e proce s s of ide ntifying the s pe cific a mino a cid a t e a ch pos ition in the pe ptide cha in, be ginning a t the N-te rmina l e nd. Phe nylis othiocya na te , known a s Edma n re a ge nt, is us e d to la be l the a mino-te rmina l re s idue unde r mildly a lka line conditions (Figure 2.4). The re s ulting phe nylthiohyda ntoin (P TH) de riva tive introduce s a n ins ta bility in the N-te rmina l pe ptide bond s uch tha t it ca n be hydrolyze d without cle a ving the othe r pe ptide bonds . The ide ntity of the a mino a cid de riva tive ca n the n be de te rmine d. Edma n re a ge nt ca n be a pplie d re pe a te dly to the s horte ne d pe ptide obta ined in e a ch pre vious cycle . The proce s s is now a utoma te d.

S e parate d amino ac ids Ninhydrin pump

Re ac tio n c o il

Pho to me te r

D. Cle avag e o f the po lype ptide into s malle r frag me nts Ma ny polype ptide s ha ve a prima ry s tructure compos e d of more tha n 100 a mino a cids . S uch mole cule s ca nnot be s e que nce d dire ctly from e nd to e nd. Howe ve r, the s e la rge mole cule s ca n be cle a ve d a t s pe cific s ite s a nd the re s ulting fra gme nts s e que nce d. By us ing more tha n one cle a ving a ge nt (e nzyme s a nd/or che mica ls ) on s e pa ra te s a mple s of the purifie d polype ptide , ove rla pping fra gme nts ca n be ge ne ra te d tha t pe rmit the prope r orde ring of the s e que nce d fra gme nts , the re by providing a comple te a mino a cid s e que nce of the la rge polype ptide (Figure 2.5). Enzyme s tha t hydrolyze pe ptide bonds a re te rme d pe ptida s e s (prote a s e s ). [Note : Exope ptida s e s cut a t the e nds of prote ins a nd a re divide d into a minope ptida s e s a nd ca rboxype ptida s e s . Ca rboxype ptida s e s a re us e d in de te rmining the C-te rmina l a mino a cid. Endope ptida s e s cle a ve within a prote in.]

Lig ht s o urc e

Strip-c hart re c o rde r o r c o mpute r

Fig ure 2.3 De te rmina tion of the a mino a cid compos ition of a polype ptide us ing a n a mino a cid a na lyze r.

E. Determination of a protein’s primary s tructure by DNA s equencing The s e que nce of nucle otide s in a prote in-coding re gion of the DNA s pe cifie s the a mino a cid s e que nce of a polype ptide . The re fore , if the nucle otide s e que nce ca n be de te rmine d, it is pos s ible , from knowle dge of the ge ne tic code (s e e p. 432), to tra ns la te the s e que nce of nucle otide s into the corre s ponding a mino a cid s e que nce of tha t polype ptide . This indire ct proce s s , a lthough routine ly us e d to

1 O H2 N CH C CH3

N-te rminal alanine

Lys

His

Le u

Pe ptide

Arg

2

Labe ling O HN CH C

COOH

CH3

S C N

Phe nylis o thio c yanate

S C NH

Lys

His

Le u

Arg

Labe le d pe ptide

Re le as e o f amino ac id de rivative by ac id hydro lys is COOH

H2 N Lys

His

Le u

Arg

COOH

S ho rte ne d pe ptide

+

S

N C NH C O CH CH3

PTH-alanine

Fig ure 2.4 De te rmina tion of the a mino (N)-te rmina l re s idue of a polype ptide by Edma n de gra da tion. P TH = phe nylthiohyda ntoin.

16

2. S tructure of P rote ins

P e ptide of unknown s e que nce

1

1. Cle ave with tryp s in at lys ine and arg inine 2. De te rmine s e que nc e o f pe ptide s us ing the Edman me tho d Pe ptide A

Pe ptide B A A B B C C

What is the c o rre c t o rde r?

B C A C A B

Pe ptide C

C? B? C? A? B? A?

P e ptide of unknown s e que nce

2

1. Cle ave with c yano g e n bro mide at me thio nine 2. De te rmine s e que nc e o f pe ptide s us ing the Edman me tho d

Pe ptide X

Pe ptide Y

Orig inal s e que nc e o f pe ptide

Fig ure 2.5 Ove rla pping of pe ptide s produce d by the a ction of tryps in a nd cya noge n bromide .

S ide c hains o f amino ac ids e xte nd o utward

Intrac hain hydro g e n bo nd

N C O H C O N C CH O

N H

C

C

R

N C H N H O C

R

C

C

R

O O

C

obta in the a mino a cid s e que nce s of prote ins , ha s the limita tions of not be ing a ble to pre dict the pos itions of dis ulfide bonds in the folde d cha in a nd of not ide ntifying a ny a mino a cids tha t a re modifie d a fte r the ir incorpora tion into the polype ptide (pos ttra ns la tiona l modifica tion, s e e p. 443). The re fore , dire ct prote in s e que ncing is a n e xtre me ly importa nt tool for de te rmining the true cha ra cte r of the prima ry s e que nce of ma ny polype ptide s .

III. S ECONDARY S TRUCTURE OF PROTEINS The polype ptide ba ckbone doe s not a s s ume a ra ndom thre e -dime ns iona l s tructure but, ins te a d, ge ne ra lly forms re gula r a rra nge me nts of a mino a cids tha t a re loca te d ne a r e a ch othe r in the line ar s e que nce . The s e a rra nge me nts a re te rme d the s e conda ry s tructure of the polype ptide . The α -he lix, β-s he e t, a nd β-be nd (β-turn) a re e xa mple s of s e conda ry s tructure s commonly e ncounte re d in prote ins . [Note : The colla ge n α -cha in he lix, a nothe r e xa mple of s e conda ry s tructure , is dis cus s e d on p. 45.] A. α-He lix S e ve ra l diffe re nt polype ptide he lice s a re found in na ture , but the α -he lix is the mos t common. It is a s pira l s tructure , cons is ting of a tightly pa cke d, coile d polype ptide ba ckbone core , with the s ide cha ins of the compone nt a mino a cids e xte nding outwa rd from the ce ntra l a xis to a void inte rfe ring s te rica lly with e a ch othe r (Figure 2.6). A ve ry dive rs e group of prote ins conta ins α -he lice s . For e xa mple , the ke ra tins a re a fa mily of clos e ly re la te d, fibrous prote ins whos e s tructure is ne a rly e ntire ly α -he lica l. The y a re a ma jor compone nt of tis s ue s s uch a s ha ir a nd s kin, a nd the ir rigidity is de te rmine d by the numbe r of dis ulfide bonds be twe e n the cons titue nt polype ptide cha ins . In contra s t to ke ra tin, myoglobin, whos e s tructure is a ls o highly α -he lica l, is a globula r, fle xible mole cule (s e e p. 26). 1. Hydro g e n bo nds : An α -he lix is s ta bilize d by e xte ns ive hydroge n

bonding be twe e n the pe ptide -bond ca rbonyl oxyge ns a nd a mide hydroge ns tha t a re pa rt of the polype ptide ba ckbone (s e e Figure 2.6). The hydroge n bonds e xte nd up a nd a re pa ra lle l to the s pira l from the ca rbonyl oxyge n of one pe ptide bond to the – NH – group of a pe ptide linka ge four re s idue s a he a d in the polype ptide . This ins ure s tha t a ll but the firs t a nd la s t pe ptide bond compone nts a re linke d to e a ch othe r through intra cha in hydroge n bonds . Hydroge n bonds a re individua lly we a k, but the y colle ctive ly s e rve to s ta bilize the he lix.

C NC H C

2. Amino ac ids pe r turn: Ea ch turn of a n α -he lix conta ins 3.6 a mino R

N H

a cids . Thus , a mino a cid re s idue s s pa ce d thre e or four re s idue s a pa rt in the prima ry s e que nce a re s pa tia lly clos e toge the r whe n folde d in the α -he lix. 3. Amino ac ids that dis rupt an α-he lix: P roline dis rupts a n α -he lix

Fig ure 2.6 α -He lix s howing pe ptide ba ckbone .

be ca us e its s e conda ry a mino group is not ge ome trica lly compa tible with the right-ha nde d s pira l of the α -he lix. Ins te a d, it ins e rts a kink in the cha in, which inte rfe re s with the s mooth, he lica l s tructure . La rge numbe rs of cha rge d a mino a cids (for e xa mple ,

III. S e conda ry S tructure of P rote ins gluta ma te , a s pa rta te , his tidine , lys ine , a nd a rginine ) a ls o dis rupt the he lix by forming ionic bonds or by e le ctros ta tica lly re pe lling e a ch othe r. Fina lly, a mino a cids with bulky s ide cha ins , s uch a s tryptopha n, or a mino a cids , s uch a s va line or is ole ucine , tha t bra nch a t the β-ca rbon (the firs t ca rbon in the R group, ne xt to the α -ca rbon) ca n inte rfe re with forma tion of the α -he lix if the y a re pre s e nt in la rge numbe rs .

17

A Hydro g e n bo nds be twe e n c hains

R-C-H

B. β-S he e t

C H

The β-s he e t is a nothe r form of s e conda ry s tructure in which a ll of the pe ptide bond compone nts a re involve d in hydroge n bonding (Figure 2.7A). The s urfa ce s of β-s he e ts a ppe a r “ple a te d,” a nd the s e s tructure s a re , the re fore , ofte n ca lle d β-ple a te d s he e ts . Whe n illus tra tions a re ma de of prote in s tructure , β-s tra nds a re ofte n vis ua lize d a s broa d a rrows (Figure 2.7B).

O

H C H N C

N

H C-R

O

R-C H N H

C H

O C

1. Co mparis o n o f a β-s he e t and an α-he lix: Unlike the α -he lix,

β-s he e ts a re compos e d of two or more pe ptide cha ins (β-s tra nds ), o r s e g m e n ts o f p o lype p tid e c h a in s , wh ic h a re a lm o s t fu lly e xte nde d. Note a ls o tha t the hydroge n bonds a re pe rpe ndicula r to the polype ptide ba ckbone in β-s he e ts (s e e Figure 2.7A). 2. Paralle l and antiparalle l s he e ts : A β-s he e t ca n be forme d from

two or more s e pa ra te polype ptide cha ins or s e gme nts of polype ptide cha ins tha t a re a rra nge d e ithe r a ntipa ra lle l to e a ch othe r (with the N-te rmina l a nd C-te rmina l e nds of the β-s tra nds a lte rna ting a s s hown in Figure 2.7B) or pa ra lle l to e a ch othe r (with a ll the N-te rmini of the β-s tra nds toge the r a s s hown in Figure 2.7C). Whe n the hydroge n bonds a re forme d be twe e n the polype ptide ba ckbone s of s e pa ra te polype ptide cha ins , the y a re te rme d inte rcha in bonds . A β-s he e t ca n a ls o be forme d by a s ingle polype ptide cha in folding ba ck on its e lf (s e e Figure 2.7C). In this ca s e , the hydroge n bonds a re intra cha in bonds . In globula r prote ins , β-s he e ts a lwa ys ha ve a right-ha nde d curl, or twis t, whe n vie we d a long the polype ptide ba ckbone . [Note : Twis te d β-s he e ts ofte n form the core of globula r prote ins .]

Po lype ptide c hains almo s t fully e xte nde d

B N-te rminal C-te rminal C-te rminal N-te rminal Antiparalle l β-ple ate d s he e t

The α -he lix a nd β-s he e t s tructure s provide ma xima l hydroge n bonding for pe ptide bond compone nts within the inte rior of polype ptide s .

C. β-Be nds (re ve rs e turns , β-turns ) β-Be nds re ve rs e the dire ction of a polype ptide cha in, helping it form a compa ct, globula r s ha pe . The y a re us ua lly found on the s urfa ce of prote in mole cule s a nd ofte n include cha rge d re s idue s . [Note : β-Be nds we re give n this na me be ca us e the y ofte n conne ct s ucce s s ive s tra nds of a ntipa ra lle l β-s he e ts .] β-Be nds a re ge ne ra lly compos e d of four a mino a cids , one of which ma y be proline , the a mino a cid tha t ca us e s a kink in the polype ptide cha in. Glycine , the a mino a cid with the s ma lle s t R group, is a ls o fre que ntly found in β-be nds . β-Be nds a re s ta bilize d by the forma tion of hydroge n a nd ionic bonds .

C

N-terminal

C-te rminal Paralle l β-ple ate d s he e t

Fig ure 2.7 A. S tructure of a β-s he e t. B. An a ntipa ra lle l β-s he e t with the β-s tra nds re pre s e nte d a s broa d a rrows . C. A pa ra lle l β-s he e t forme d from a s ingle polype ptide cha in folding ba ck on its e lf.

18

2. S tructure of P rote ins

α-α (He lix-lo o p-he lix)

β-α-β

β-Me ande r

β-Barre l

Fig ure 2.8 S ome common s tructura l motifs involving α -he lice s a nd β-s he e ts . The na me s de s cribe the ir s che ma tic a ppe a ra nce . D. No nre pe titive s e c o ndary s truc ture Approxima te ly one ha lf of a n a ve ra ge globula r prote in is orga nize d into re pe titive s tructure s , s uch a s the α -he lix a nd β-s he e t. The re ma inde r of the polype ptide cha in is de s cribe d a s ha ving a loop or coil conforma tion. The s e nonre pe titive s e conda ry s tructure s a re not ra ndom, but ra the r s imply ha ve a le s s re gula r s tructure tha n thos e de s cribe d a bove . [Note : The te rm “ra ndom coil” re fe rs to the dis orde re d s tructure obta ine d whe n prote ins a re de na ture d (s e e p. 20).] H O N C C H CH2 SH Two c ys te ine Po lype ptide re s idue s bac kbo ne SH H CH2 N C C H O

Oxida nt (for e xa mple , O 2 )

H O N C C H CH2

E. S upe rs e c o ndary s truc ture s (mo tifs ) Globula r prote ins a re cons tructe d by combining s e conda ry s tructura l e le me nts (tha t is , α-he lice s , β-s he e ts , a nd coils ), producing s pe cific ge ome tric pa tte rns or motifs . The s e form prima rily the core (inte rior) re gion of the mole cule . The y a re conne cte d by loop re gions (for e xa mple , β-be nds ) a t the s urfa ce of the prote in. S upe rs e conda ry s tructure s a re us ua lly produce d by the clos e pa cking of s ide cha ins from a dja ce nt s e conda ry s tructura l e le me nts . Thus , for e xa mple , α-he lice s a nd β-s he e ts tha t a re a dja ce nt in the a mino a cid s e que nce a re a ls o us ua lly (but not a lwa ys ) a dja ce nt in the fina l, folde d prote in. Some of the more common motifs a re illus tra te d in Figure 2.8.

Motifs ma y be a s s ocia te d with pa rticula r functions . P rote ins tha t bind to DNA conta in a limite d numbe r of motifs . The he lix-loop-he lix motif is a n e xa mple found in a numbe r of prote ins tha t function a s tra ns cription fa ctors (s e e p. 450).

IV. TERTIARY S TRUCTURE OF GLOBULAR PROTEINS Dis ulfide bo nd

Fig ure 2.9 Forma tion of a dis ulfide bond by the oxida tion of two cys te ine re s idue s , producing one cys tine re s idue .

The prima ry s tructure of a polype ptide cha in de te rmine s its te rtia ry s tructure . “Te rtia ry” re fe rs both to the folding of doma ins (the ba s ic units of s tructure a nd function, s e e dis cus s ion be low), a nd to the fina l a rra nge me nt of doma ins in the polype ptide . The s tructure of globula r prote ins in a que ous s olution is compa ct, with a high de ns ity (clos e pa cking) of the a toms in the core of the mole cule . Hydrophobic s ide cha ins a re burie d in the inte rior, whe re a s hydrophilic groups a re ge ne ra lly found on the s urfa ce of the mole cule .

IV. Te rtia ry S tructure of Globula r P rote ins

19

A. Do mains H H O N C C H C CH3 C H 2 Is o le uc ine

Doma ins a re the funda me nta l functiona l a nd thre e -dime ns iona l s tructura l units of polype ptide s . P olype ptide cha ins tha t a re gre a te r tha n 200 a mino a cids in le ngth ge ne ra lly cons is t of two or more doma ins . The core of a doma in is built from combina tions of s upe rs e conda ry s tructura l e le me nts (motifs ). Folding of the pe ptide cha in within a doma in us ua lly occurs inde pe nde ntly of folding in othe r doma ins . The re fore , e a ch doma in ha s the cha ra cte ris tics of a s ma ll, compa ct globula r prote in tha t is s tructura lly inde pe nde nt of the othe r doma ins in the polype ptide cha in.

CH3

B. Inte rac tio ns s tabilizing te rtiary s truc ture The unique thre e -dime ns iona l s tructure of e a ch polype ptide is de te rmine d by its a mino a cid s e que nce . Inte ra ctions be twe e n the a mino a cid s ide cha ins guide the folding of the polype ptide to form a compa ct s tructure . The following four type s of inte ra ctions coope ra te in s ta bilizing the te rtia ry s tructure s of globula r prote ins . 1. Dis ulfide bo nds : A dis ulfide bond is a cova le nt linka ge forme d

from the s ulfhydryl group (–S H) of e a ch of two cys te ine re s idue s to produce a cys tine re s idue (Figure 2.9). The two cys te ine s ma y be s e pa ra te d from e a ch othe r by ma ny a mino a cids in the prima ry s e que nce of a polype ptide or ma y e ve n be loca te d on two diffe re nt polype ptide cha ins . The folding of the polype ptide cha in(s ) brings the cys te ine re s idue s into proximity a nd pe rmits cova le nt bonding of the ir s ide cha ins . A dis ulfide bond contribute s to the s ta bility of the thre e -dime ns iona l s ha pe of the prote in mole cule a nd pre ve nts it from be coming de na ture d in the e xtra ce llula r e nvironme nt. For e xa mple , ma ny dis ulfide bonds a re found in prote ins s uch a s immunoglobulins tha t a re s e cre te d by ce lls .

Hydro pho bic inte rac tio ns

Fig ure 2.10 Hydrophobic inte ra ctions be twe e n a mino a cids with nonpola r s ide cha ins .

Glutamate

As partate

H H O N C C

H H O N C C

CH2 CH2

2. Hydro pho bic inte rac tio ns : Amino a cids with nonpola r s ide cha ins

te nd to be loca te d in the inte rior of the polype ptide mole cule , whe re the y a s s ocia te with othe r hydrophobic a mino a cids (Figure 2.10). In contra s t, a mino a cids with pola r or cha rge d s ide cha ins te nd to be loca te d on the s urfa ce of the mole cule in conta ct with the pola r s olve nt. [Note : Re ca ll tha t prote ins loca te d in nonpola r (lipid) e nvironme nts , s uch a s a me mbra ne , e xhibit the re ve rs e a rra nge me nt (s e e Figure 1.4, p. 4).] In e a ch ca s e , a s e gre ga tion of R groups occurs tha t is e ne rge tica lly mos t fa vora ble . 3. Hydro g e n bo nds : Amino a cid s ide cha ins conta ining oxyge n- or

nitroge n-bound hydroge n, s uch a s in the a lcohol groups of s e rine a nd thre onine , ca n form hydroge n bonds with e le ctron-rich a toms , s uch a s the oxyge n of a ca rboxyl group or ca rbonyl group of a pe ptide bond (Figure 2.11; s e e a ls o Figure 1.6, p. 4). Forma tion of hydroge n bonds betwe e n pola r groups on the s urfa ce of prote ins a nd the a que ous s olve nt e nha nce s the s olubility of the prote in. 4. Io nic inte rac tio ns : Ne ga tive ly cha rge d groups , s uch a s the ca r-

boxyla te group (– COO – ) in the s ide cha in of a s pa rta te or gluta ma te , ca n inte ra ct with pos itive ly cha rge d groups s uch a s the a mino group (– NH3 +) in the s ide cha in of lys ine (s e e Figure 2.11).

CH2 C O

C

O–

O–

O

+NH

3

N C C

CH2 C CH2 CH2 H CH2 N C C

H O

H O

H O H CH2

S e rine

Hydro g e n bo nd

Lys ine

Io nic bo nd

Fig ure 2.11 Inte ra ctions of s ide cha ins of a mino a cids through hydroge n bonds a nd ionic bonds (s a lt bridge s ).

20

2. S tructure of P rote ins C. Pro te in fo lding

1

Fo rmatio n o f s e c o ndary s truc ture s

Inte ra ctions be twe e n the s ide cha ins of a mino a cids de te rmine how a long polype ptide cha in folds into the intrica te thre e -dime ns iona l s ha pe of the functiona l prote in. P rote in folding, which occurs within the ce ll in s e conds to minute s , involve s nonra ndom, orde re d pa thwa ys . As a pe ptide folds , s e conda ry s tructure s form drive n by the hydrophobic e ffe ct (tha t is , hydrophobic groups come toge the r a s wa te r is re le a s e d). The s e s ma ll s tructure s combine to form la rge r s tructure s . Additiona l e ve nts s ta bilize s e conda ry s tructure a nd initia te forma tion of te rtia ry s tructure . In the la s t s ta ge , the pe ptide a chie ve s its fully folde d, na tive (functiona l) form cha ra cte rize d by a low-e ne rgy s ta te (Figure 2.12). [Note : S ome biologica lly a ctive prote ins or s e gme nts the re of la ck a s ta ble te rtia ry s tructure . The y a re re fe rre d to a s “intrins ica lly dis orde re d” prote ins .] D. De naturatio n o f pro te ins

2

Fo rmatio n o f do mains

P rote in de na tura tion re s ults in the unfolding a nd dis orga niza tion of a prote in’s s e conda ry a nd te rtia ry s tructure s without the hydrolys is of pe ptide bonds . De na turing a ge nts include he a t, orga nic s olve nts , s trong a cids or ba s e s , de te rge nts , a nd ions of he a vy me ta ls s uch a s le a d. De na tura tion ma y, unde r ide a l conditions , be re ve rs ible , s uch tha t the prote in re folds into its origina l na tive s tructure whe n the de na turing a ge nt is re move d. Howe ve r, mos t prote ins , once de na ture d, re ma in pe rma ne ntly dis orde re d. De na ture d prote ins a re ofte n ins oluble a nd pre cipita te from s olution. E. Ro le o f c hape ro ne s in pro te in fo lding

3

Fo rmatio n o f final pro te in mo no me r

Fig ure 2.12 S te ps in prote in folding (s implifie d).

The informa tion ne e de d for corre ct prote in folding is conta ine d in the prima ry s tructure of the polype ptide . Howe ve r, mos t prote ins whe n de na ture d do not re s ume the ir na tive conforma tions e ve n unde r fa vora ble e nvironme nta l conditions . This is be ca us e , for ma ny prote ins , folding is a fa cilita te d proce s s tha t re quire s a s pe cia lize d group of prote ins , re fe rre d to a s “mole cula r cha pe rone s ,” a nd a de nos ine triphos pha te hydrolys is . The cha pe rone s , a ls o known a s “he a t s hock prote ins ” (Hs p), inte ra ct with a polype ptide a t va rious sta ge s during the folding proce s s . S ome cha pe rone s bind hydrophobic re gions of a n e xte nde d polype ptide a nd a re importa nt in ke e ping the prote in unfolde d until its s ynthe s is is comple te d (for e xa mple , Hs p70). Othe rs form ca ge -like ma cromole cula r s tructure s compos e d of two s ta cke d rings . The pa rtia lly folde d prote in e nte rs the ca ge , binds the ce ntra l ca vity through hydrophobic inte ra ctions , folds , a nd is re le a s e d (for e xa mple , mitochondria l Hs p60). [Note : Ca ge -like cha pe rone s a re some time s re fe rre d to a s “cha pe ronins .”] Cha pe rone s, the n, fa cilita te corre ct prote in folding by binding to a nd s ta bilizing e xpos e d, a ggre ga tion-prone hydrophobic re gions in na s ce nt (a nd de na ture d) polype ptide s , pre ve nting pre ma ture folding.

V. QUATERNARY S TRUCTURE OF PROTEINS Ma ny prote ins cons is t of a s ingle polype ptide cha in a nd a re de fine d a s monome ric prote ins . Howe ve r, othe rs ma y cons is t of two or more polype ptide cha ins tha t ma y be s tructura lly ide ntica l or tota lly unre la te d. The a rra nge me nt of the s e polype ptide s ubunits is ca lle d the qua te rna ry s tructure of the prote in. S ubunits a re he ld toge the r prima rily by non-

VI. P rote in Mis folding cova le nt inte ra ctions (for e xa mple , hydroge n bonds , ionic bonds , a nd hydrophobic inte ra ctions ). S ubunits ma y e ithe r function inde pe nde ntly of e a ch othe r or ma y work coope ra tive ly, a s in he moglobin, in which the binding of oxyge n to one s ubunit of the te tra me r incre a s e s the a ffinity of the othe r s ubunits for oxyge n (s e e p. 29).

21

A Amylo id pre c urs o r pro te in

Extrac e llular

Is oforms a re prote ins tha t pe rform the s a me function but ha ve diffe re nt prima ry s tructure s . The y ca n a ris e from diffe re nt ge ne s or from tis s ue -s pe cific proce s s ing of the product of a s ingle ge ne . If the prote ins function a s e nzyme s , the y a re re fe rre d to a s is ozyme s (s e e p. 65).

Enzymic c le avag e (β -s e c re ta s e )

Ce ll me mbrane

Enzymic c le avag e (γ -s e c re ta s e )

Intrac e llular

VI. PROTEIN MIS FOLDING

B Prote in folding is a comple x proce s s tha t ca n s ome time s re s ult in imprope rly folde d mole cule s . The s e mis folde d prote ins a re us ua lly ta gge d a nd de gra de d within the ce ll (s e e p. 444). Howe ve r, this qua lity control s ys te m is not pe rfe ct, a nd intra ce llula r or e xtra cellula r a ggre ga te s of mis folde d prote ins ca n a ccumula te , pa rticula rly a s individua ls a ge . De pos its of mis folde d prote ins a re a s s ocia te d with a numbe r of dis e a s e s .

Amylo id Aβ Ce ll me mbrane

A. Amylo id dis e as e s Mis folding of prote ins ma y occur s ponta ne ous ly or be ca us e d by a muta tion in a pa rticula r ge ne , which the n produce s a n a lte re d prote in. In a ddition, s ome a ppa re ntly norma l prote ins ca n, a fte r a bnorma l prote olytic cle a va ge , ta ke on a unique conforma tiona l s ta te tha t le a ds to the forma tion of long, fibrilla r prote in a s s e mblie s cons is ting of β-ple a te d s he e ts . Accumula tion of the s e ins oluble , sponta ne ous ly a ggre ga ting prote ins , ca lle d a myloids , ha s be e n implica te d in de ge ne ra tive dis e a s e s s uch a s P a rkins on a nd Huntington a nd pa rticula rly in the a ge -re la te d ne urode ge ne ra tive dis orde r, Alzhe ime r dis e a s e . The domina nt compone nt of the a myloid pla que tha t a ccumula te s in Alzhe ime r dis e a s e is a myloid β (Aβ), a n e xtra ce llula r pe ptide conta ining 40–42 a mino a cid re s idue s . X-ra y crys ta llogra phy a nd infra re d s pe ctros copy de mons tra te a cha ra cte ris tic β-ple a te d s he e t conforma tion in nonbra nching fibrils . This pe ptide , whe n a ggre ga te d in a β-ple a te d s he e t configura tion, is ne urotoxic a nd is the ce ntra l pa thoge nic e ve nt le a ding to the cognitive impa irme nt cha ra cte ris tic of the dis e a s e . The Aβ tha t is de pos ite d in the bra in in Alzhe ime r dis e a s e is de rive d by e nzymic cle a va ge s (by s e cre ta s e s ) from the la rge r a myloid pre curs or prote in, a s ingle tra ns me mbra ne prote in e xpre s s e d on the ce ll s urfa ce in the bra in a nd othe r tis s ue s (Figure 2.13). The Aβ pe ptide s a ggre ga te , ge ne ra ting the a myloid tha t is found in the bra in pa re nchyma a nd a round blood ve s s e ls . Mos t ca s e s of Alzhe ime r dis e a s e a re not ge ne tica lly ba s e d, a lthough a t le a s t 5% of ca s e s a re fa milia l. A s e cond biologic fa ctor involve d in the de ve lopme nt of Alzhe ime r dis e a s e is the a ccumula tion of ne urofibrilla ry ta ngle s ins ide ne urons . A ke y compone nt of the s e ta ngle d fibe rs is a n a bnorma l form (hype rphos phoryla te d a nd ins oluble ) of the ta u (τ ) prote in, which, in its he a lthy ve rs ion, he lps in the a s s e mbly of the microtubula r s tructure . The de fe ctive τ a ppe a rs to block the a ctions of its norma l counte rpa rt.

S po ntane o us ag g re g atio n to fo rm ins o luble fibrils o f β-ple ate d s he e ts

C

Mo de l o f amylo id fibrils

Pho to mic ro g raph o f amylo id plaque s in a s e c tio n o f te mpo ral c o rte x fro m a patie nt with Alzhe ime r dis e as e

Fig ure 2.13 Forma tion of a myloid pla que s found in Alzhe ime r dis e a s e (AD). [Note : Muta tions to pre s e nilin, the ca ta lytic s ubunit of γ-s e cre ta s e , a re the mos t common ca us e of fa milia l AD.]

22

2. S tructure of P rote ins B. Prio n dis e as e s

1

Inte rac tio n o f the infe c tio us PrP mo le c ule with a no rmal PrP c aus e s the no rmal fo rm to fo ld into the infe c tio us fo rm.

No ninfe c tio us PrP C (c o ntains α-he lix)

Infee c tio us PrP S c (c o ntains t i β-s β-s he h e tts )

Infe c tio us PrP S c (c o ntains β-s he e ts )

2

The s e two mo le c ule s dis s o c iate and c o nve rt two additio nal no ninfe c tio us PrP mo le c ule s to the infe c tio us fo rm.

The prion prote in (P rP ) ha s be e n s trongly implica ted a s the ca us a tive a ge nt of tra ns mis s ible s pongiform e nce pha lopa thie s (TSEs ), including Cre utzfe ldt-J a kob dis e a s e in huma ns , s cra pie in s he ep, a nd bovine spongiform e nce pha lopa thy in ca ttle (popula rly ca lle d “ma d cow” dis e a s e ). Afte r a n e xte ns ive s e rie s of purifica tion proce dure s , s cie ntis ts we re s urpris e d to find tha t the infe ctivity of the a ge nt ca us ing s cra pie in s he e p wa s a s s ocia te d with a s ingle prote in s pe cie s tha t wa s not comple xe d with de te cta ble nucle ic a cid. This infe ctious prote in is de s igna te d P rP S c (S c = s cra pie ). It is highly re s is ta nt to prote olytic de gra da tion a nd te nds to form ins oluble a ggre ga te s of fibrils , s imilar to the a myloid found in s ome othe r dis e a s e s of the bra in. A noninfe ctious form of PrP C (C = ce llula r), e ncode d by the s a me ge ne a s the infe ctious a ge nt, is pre s e nt in norma l ma mma lian bra ins on the s urfa ce of ne urons a nd glia l ce lls . Thus , P rP C is a hos t prote in. No prima ry s tructure diffe re nce s or a lte rna te pos ttra ns la tiona l modifica tions ha ve be e n found be twe e n the norma l a nd the infe ctious forms of the prote in. The ke y to be coming infe ctious a ppare ntly lie s in cha nge s in the thre e -dime ns iona l conforma tion of P rP C . It ha s be e n obs e rve d tha t a numbe r of α -he lice s pre s e nt in noninfe ctious P rP C a re re pla ce d by β-s he e ts in the infe ctious form (Figure 2.14). It is presuma bly this conforma tiona l diffe re nce tha t confe rs re la tive re sis ta nce to prote olytic de gra da tion of infe ctious prions a nd pe rmits the m to be distinguis he d from the norma l P rP C in infe cte d tis s ue . The infe ctive a ge nt is , thus , a n a lte re d ve rs ion of a norma l prote in, which a cts a s a “te mpla te ” for conve rting the norma l prote in to the pa thoge nic conforma tion. The TSEs a re inva ria bly fata l, a nd no tre a tme nt is curre ntly a va ila ble tha t ca n a lte r this outcome.

VII. CHAPTER S UMMARY

No ninfe c tio us PrP C (c o ntains α-he lix)

3

No ninfe c tio us PrP C (c o ntains α-he lix)

This re s ults in an e xpo ne ntial inc re as e o f the infe c tio us fo rm.

Fig ure 2.14 One propos e d me cha nis m for multiplica tion of infe ctious prion a ge nts . P rP = prion prote in; P rP c = prion prote in ce llula r; P rP S c = prion prote in s cra pie .

Ce ntra l to unde rs ta nding prote in s tructure is the conce pt of the native co nformation (Figure 2.15), which is the functiona l, fully folded prote in structure (for example, an active enzyme or structural protein). The unique three-dimensional structure of the native conformation is determined by its primary s tructure, that is, its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide chain to form s econdary, tertiary, and (sometimes) quaternary structures, which cooperate in stabilizing the native conformation of the protein. In addition, a s pe cia lize d group of prote ins na me d c hape ro ne s is re quire d for the proper folding of many species of proteins. Protein denaturation results in the unfolding and disorganization of the protein’s structure, which are not accompanied by hydrolysis of peptide bonds. Denaturation may be reversible or, more commonly, irreversible. Disease can occur when an apparently normal protein assumes a conformation that is cytotoxic, as in the case of Alzheimer disease and the trans mis s ible s pongiform encephalo pathie s (TS Es ), including Cre utzfe ldt-Jako b dis e as e . In Alzhe ime r dis eas e, normal proteins, after abnormal chemical processing, take on a unique conformational state that leads to the formation of neurotoxic amyloid β peptide (Aβ) assemblies consisting of β-pleated sheets. In TSEs, the infective agent is an altered version of a normal prion protein that acts as a “template” for converting normal protein to the pathogenic conformation.

VII. Cha pte r S umma ry

23

Hie rarc hy o f pro te in s truc ture compos e d of

Primary is

contributes to

s e que nce of a mino a cids ca n be

Fibro us or g lo bular

leads to Chape ro ne s

α -He lix β-S he e t β-Be nds (re ve rs e turns )

cons is ts of

Nonre pe titive s tructure s

Secondary is regular arrangements contributes to of amino acids located near each other in primary structure

folding a s s is te d by

Supersecondary structures

Na t ive de te rmine s c o n fo rm a t io n

leads to

For e xa mple : Ca ta lys is P rote ction Re gula tion S igna l tra ns duction S tora ge S tructure Tra ns port

• • • • • • •

Hydrophobic inte ra ctions Hydroge n bonds

s ta bilize d by

Ele ctros ta tic inte ra ctions Dis ulfide bonds

Te rtiary is the thre e contribute s to dime ns iona l s ha pe of the folde d cha in

may lead to

Hydrophobic inte ra ctions Hydroge n bonds

stabilized by

Ele ctros ta tic inte ra ctions

Bio lo g ic func tio n

unfolding ca us e d by s ome ma y re ga in

Quate rnary is the a rra nge me nt may contribute to of polype ptide s ubunits in the prote in

ca n form

De naturants For e xa mple : Ure a Extre me s of pH, te mpe ra ture Orga nic s olve nts

• • •

le a d to Lo s s o f s e c o ndary and te rtiary s truc ture le a ds to Lo s s o f func tio n

Cre utzfe ldt-Jako b dis e as e

le a d to

P rions

le a ds to Alte re d folding

Alzhe ime r dis e as e (the mos t common ca us e of de me ntia in olde r a dults )

le a d to

le a ds to

mos t prote ins ca nnot re fold upon re mova l of de na tura nt

Amyloid prote ins

Fig ure 2.15 Ke y conce pt ma p for prote in s tructure .

Irre ve rs ible de naturatio n

24

2. S tructure of P rote ins

S tudy Que s tio ns Choos e the ONE be s t a ns we r. 2.1 Which one of the following s ta te me nts conce rning prote in s tructure is corre ct? A. P rote ins cons is ting of one polype ptide ha ve qua te rna ry s tructure tha t is s ta bilize d by cova le nt bonds . B. The pe ptide bonds tha t link a mino a cids in a prote in mos t commonly occur in the cis configura tion. C. Th e fo rm a tio n o f a d is u lfid e b o n d in a p ro te in re quire s the pa rticipa ting cys te ine re s idue s to be a dja ce nt in the prima ry s tructure . D. The de na tura tion of prote ins le a ds to irre ve rs ible los s of s e conda ry s tructura l e le me nts s uch a s the α -he lix. E. The prima ry driving force for prote in folding is the hydrophobic e ffe ct. 2.2 A pa rticula r point muta tion re s ults in dis ruption of the α -he lica l s tructure in a s e gme nt of the muta nt prote in. The mos t like ly cha nge in the prima ry s tructure of the muta nt prote in is : A. B. C. D.

gluta ma te to a s pa rta te . lys ine to a rginine . me thionine to proline . va line to a la nine .

2.3 In compa ring the α -he lix to the β-s he e t, which s ta te me nt is corre ct only for the β-s he e t? A. Exte ns ive hydroge n bonds be twe e n the ca rbonyl oxyge n (C=O) a nd the a mide hydroge n (N-H) of the pe ptide bond a re forme d. B. It ma y be found in typica l globula r prote ins . C. It is s ta bilize d by inte rcha in hydroge n bonds . D. it is a n e xa mple of s e conda ry s tructure . E. It ma y be found in s upe rs e conda ry s tructure s . 2.4 An 80-ye a r-old ma n pre s e nte d with impa irme nt of highe r inte lle ctua l function a nd a lte ra tions in mood a nd be ha vior. His fa mily re porte d progre s s ive dis orie nta tion a nd me mory los s ove r the la s t 6 months . The re is no fa mily his tory of de me ntia . The pa tie nt wa s te ntative ly dia gnos e d with Alzhe ime r dis e a s e . Which one of the following be s t de s cribe s Alzhe ime r dis e a s e ? A. It is a s s ocia te d with β-a myloid, a n a bnorma l prote in with a n a lte re d a mino a cid s e que nce . B. It re s ults from a ccumula tion of de na ture d prote ins tha t ha ve ra ndom conforma tions . C. It is a s s ocia te d with the a ccumula tion of a myloid pre curs or prote in. D. It is a s s ocia te d with the de pos ition of ne urotoxic a myloid β pe ptide a ggre ga te s . E. It is a n e nvironme nta lly produce d dis e a s e not influe nce d by the ge ne tics of the individua l. F. It is ca use d by the infe ctious β-she e t form of a hostce ll prote in.

Corre ct a ns we r = E. The hydrophobic e ffe ct, or the te nde ncy of nonpola r e ntitie s to a s s ocia te in a pola r e nvironme nt, is the driving force of prote in folding. Qua te rna ry s tructure re quire s more tha n one polype ptide , a nd, whe n pre s e nt, it is s ta bilize d prima rily by noncova le nt bonds . The pe ptide bond is a lmos t a lwa ys tra ns . The two cys te ine re s idue s pa rticipa ting in dis ulfide bond forma tion ma y be a gre a t dis ta nce a pa rt in the a mino a cid s e que nce of a polype ptide (or on two s e pa ra te polype ptide s ) but a re brought into clos e proximity by the thre e -dime ns iona l folding of the polype ptide . De na tura tion ma y be re ve rs ible or irre ve rs ible .

Corre ct a ns we r = C. P roline , be ca us e of its s e conda ry a mino group, is incompa tible with a n α -he lix. Gluta ma te , a s pa rta te , lys ine , a nd a rginine a re cha rge d a mino a cids , a nd va line is a bra nche d a mino a cid. Cha rge d a nd bra nche d (bulky) a mino a cids ma y dis rupt a n α -he lix.

Corre ct a nswe r = C. The β-she e t is sta bilize d by inte rcha in hydroge n bonds forme d be twe e n se pa ra te polype ptide cha ins a nd by intra cha in hydroge n bonds forme d be twe e n re gions of a s ingle polype ptide . The α -he lix, howe ve r, is s ta bilize d only by intra cha in hydroge n bonds . S ta te me nts A, B, D, a nd E a re true for both of the se s e conda ry structural e le me nts.

C o rre c t a n s we r = D. Alz h e im e r d is e a s e is a s s ocia te d with long, fibrilla r prote in a s s e mblie s cons is ting of β-ple a te d s he e ts found in the bra in a nd e ls e whe re . The dis e a s e is a s s ocia te d with a bnorma l proce s s ing of a norma l prote in. The a ccumula te d a lte re d prote in occurs in a β-ple a te d s he e t configura tion tha t is ne urotoxic. The a myloid β tha t is de pos ite d in the bra in in Alzhe ime r dis e a s e is de rive d by prote olytic cle a va ge s from the la rge r a myloid pre curs or prote in, a s ingle tra ns me mbra ne prote in e xpre s s e d on the ce ll s urfa ce in the bra in a nd othe r tis s ue s . Mos t ca s e s of Alzhe ime r dis e a s e a re s pora dic, a lthough a t le a s t 5% of ca s e s a re fa milia l. P rion dis e a s e s , s uch a s Cre utzfe ldt-J a kob, a re ca us e d by the infe ctious β-s he e t form (P rP S c ) of a hos tce ll prote in (P rP C ).

3

Glo bular Pro te ins I. OVERVIEW

A

The pre vious cha pte r de s cribe d the type s of s e conda ry a nd te rtia ry s tructure s tha t a re the bricks a nd morta r of prote in a rchite cture . By a rra nging the s e funda me nta l s tructura l e le me nts in diffe re nt combina tions , wide ly dive rs e prote ins ca n be cons tructe d tha t a re ca pa ble of va rious s pe cia lize d functions . This cha pte r e xa mine s the re la tions hip be twe e n s tructure a nd function for the clinica lly importa nt globula r he me prote ins . Fibrous s tructura l prote ins a re dis cus s e d in Cha pte r 4.

II. GLOBULAR HEMEPROTEINS He me prote ins a re a group of s pe cia lize d prote ins tha t conta in he me a s a tightly bound pros the tic group. (S e e p. 54 for a dis cus s ion of pros the tic groups .) The role of the he me group is dicta te d by the e nvironme nt cre a te d by the thre e -dime ns iona l s tructure of the prote in. For e xa mple , the he me group of a cytochrome functions a s a n e le ctron ca rrie r tha t is a lte rna te ly oxidize d a nd re duce d (s e e p. 76). In contra s t, the he me group of the e nzyme ca ta la s e is pa rt of the a ctive s ite of the e nzyme tha t ca ta lyze s the bre a kdown of hydroge n pe roxide (s e e p. 148). In he moglobin a nd myoglobin, the two mos t a bunda nt he me prote ins in huma ns , the he me group s e rve s to re ve rs ibly bind oxyge n. A. S truc ture o f he me He me is a comple x of protoporphyrin IX a nd fe rrous iron (Fe 2+ ) (Figure 3.1). The iron is he ld in the ce nte r of the he me mole cule by bonds to the four nitroge ns of the porphyrin ring. The he me Fe 2+ ca n form two a dditiona l bonds , one on e a ch s ide of the pla na r porphyrin ring. In myoglobin a nd he moglobin, one of the s e pos itions is coordina te d to the s ide cha in of a his tidine re s idue of the globin mole cule , whe re a s the othe r pos ition is a va ila ble to bind oxyge n (Figure 3.2). (S e e pp. 278 a nd 282 for a dis cus s ion of the s ynthe s is a nd de gra da tion of he me .)

B Iro n c an fo rm s ix bo nds : fo ur with po rphyrin nitro g e ns , plus two additio nal bo nds , o ne abo ve and o ne be lo w the planar po rphyrin ring .

COO COOCH2 CH2 H C C H3 C C C C N Fe HC C N H2 C C C C C C H CH H 3

C

C

CO COO OOCH H2 CH H2 C

CH3

N C C N C C CH3 C C C H CH2

Fig ure 3.1 A. He me prote in (cytochrome c). B. S tructure of he me .

25

26

3. Globula r P rote ins

Pro ximal his tidine (F8)

B

A B G

H

D

C

Fe

E

F

Oxyg e n mo le c ule (O2 )

He me He me

Dis tal his tidine (E7)

A

F He lix

E He lix

Fig ure 3.2 A. Mode l of myoglobin s howing he lice s A to H. B. S che ma tic dia gra m of the oxyge n-binding s ite of myoglobin. B. S truc ture and func tio n o f myo g lo bin Myoglobin, a he me prote in pre s e nt in he a rt a nd s ke le ta l mus cle , functions both a s a re s e rvoir for oxyge n a nd a s a n oxyge n ca rrie r tha t incre a s e s the ra te of tra ns port of oxyge n within the mus cle ce ll. [Note : Mous e myoglobin double knockouts (s e e p. 486) ha ve , s urpris ingly, a n a ppa re ntly norma l phe notype .] Myoglobin cons is ts of a s ingle polype ptide cha in tha t is s tructura lly s imila r to the individua l polype ptide cha ins of the te tra me ric he moglobin mole cule . This homology ma ke s myoglobin a us e ful mode l for inte rpre ting s ome of the more comple x prope rtie s of he moglobin. 1. α-He lic al c o nte nt: Myoglobin is a compa ct mole cule , with a pprox-

ima te ly 80% of its polype ptide cha in folde d into e ight s tre tche s of α -he lix. The s e α -he lica l re gions , la be le d A to H in Figure 3.2A, a re te rmina te d e ithe r by the pre s e nce of proline , whos e five me mbe re d ring ca nnot be a ccommoda te d in a n α -he lix (s e e p. 16) or by β-be nds a nd loops s ta bilize d by hydroge n bonds a nd ionic bonds (s e e p. 17). [Note : Ionic bonds a re a ls o te rme d e le ctros ta tic inte ra ctions or s a lt bridge s .] 2. Lo c atio n o f po lar and no npo lar amino ac id re s idue s : The inte rior

of the myoglobin mole cule is compos e d a lmos t e ntire ly of nonpola r a mino a cids . The y a re pa cke d clos e ly toge the r, forming a s tructure s ta bilize d by hydrophobic inte ra ctions be twe e n the s e clus te re d re s idue s (s e e p. 19). In contra s t, pola r a mino a cids a re loca te d a lmos t e xclus ive ly on the s urfa ce , whe re the y ca n form hydroge n bonds , both with e a ch othe r a nd with wa te r. 3. Binding o f the he me g ro up: The he me group of the myoglobin

mole cule s its in a cre vice , which is line d with nonpola r a mino a cids . Nota ble e xce ptions a re two his tidine re s idue s (Figure 3.2B). One , the proxima l his tidine (F8), binds dire ctly to the iron of he me . The s e cond, or dis ta l his tidine (E7), doe s not dire ctly inte ra ct with the he me group but he lps s ta bilize the binding of oxyge n to the fe rrous iron. The prote in, or globin, portion of myoglobin thus cre a te s a s pe cia l microe nvironme nt for the he me tha t pe rmits the re ve rs ible binding of one oxyge n mole cule (oxyge na tion). The s imulta ne ous los s of e le ctrons by the fe rrous iron (oxida tion to the fe rric form) occurs only ra re ly.

II. Globula r He me prote ins

A

27

β2

β1

α2

α1

B

Fig ure 3.3 A. S tructure of he moglobin s howing the polype ptide ba ckbone . B. S implifie d dra wing s howing the he lice s . C. S truc ture and func tio n o f he mo g lo bin He moglobin is found e xclus ive ly in re d blood ce lls (RBC), whe re its ma in function is to tra ns port oxyge n (O 2 ) from the lungs to the ca pilla rie s of the tis s ue s . He moglobin A, the ma jor he moglobin in a dults , is compos e d of four polype ptide cha ins (two α cha ins a nd two β cha ins ) he ld toge the r by noncova le nt inte ra ctions (Figure 3.3). Ea ch cha in (s ubunit) ha s s tre tche s of α -he lica l s tructure a nd a hydrophobic he me -binding pocke t s imila r to tha t de s cribe d for myoglobin. Howe ve r, the te tra me ric he moglobin mole cule is s tructura lly a nd functiona lly more comple x tha n myoglobin. For e xa mple , he moglobin ca n tra ns port H+ a nd CO 2 from the tis s ue s to the lungs a nd ca n ca rry four mole cule s of O 2 from the lungs to the ce lls of the body. Furthe rmore , the oxyge n-binding prope rtie s of he moglobin a re re gula te d by inte ra ction with a llos te ric e ffe ctors (s e e p. 29).

Obta ining O 2 from the a tmos phe re s ole ly by diffus ion gre a tly limits the s ize of orga nis ms . Circula tory s ys te ms ove rcome this , but tra ns port mole cule s s uch a s he moglobin a re a ls o re quire d be ca us e O 2 is only s lightly s oluble in a que ous s olutions s uch a s blood.

1. Quate rnary s truc ture o f he mo g lo bin: The he moglobin te tra me r

ca n be e nvis ione d a s be ing compos e d of two ide ntica l dime rs , (αβ)1 a nd (αβ)2 . The two polype ptide cha ins within e a ch dime r a re he ld tightly toge the r prima rily by hydrophobic inte ra ctions (Figure 3.4). [Note : In this ins ta nce , hydrophobic a mino a cid re s idue s a re loca lize d not only in the inte rior of the mole cule , but a ls o in a re gion on the s urfa ce of e a ch s ubunit. Multiple inte rcha in hydrophobic inte ra ctions form s trong a s s ocia tions be twe e n α-s ubunits a nd β-s ubunits in the dime rs .] In contra s t, the two dime rs a re

28

3. Globula r P rote ins

We ak io nic and hydro g e n bo nds o c c ur be twe e n αβ dime r pairs in the de o xyg e nate d s tatee .

S tro ng inte rac tio ns , primarily hydro pho bic , be twe e n α and β c hains fo rm s table αβ dime rs .

αβ

αβ

O2 αβ dime r 1

4 O2

4 O2

αβ dime r 2 O2

"T," o r taut, s truc ture o f de o xyhe mo g lo bin

O2

S o me io nic and hydro g e n bo nds be twe e n αβ dime rs are bro ke n in the o xyg e nate d s tate .

O2

"R," o r re laxe d, s truc ture o f o xyhe mo g lo bin

Fig ure 3.4 . tion of he moglobin. S che ma tic dia gra m s howing s tructura l cha nge s re s ulting from oxyge na tion a nd de oxyge na

he ld toge the r prima rily by pola r bonds . The we a ke r inte ra ctions be twe e n the dime rs a llows the m to move with re s pe ct to one othe r. This move me nt re s ults in the two dime rs occupying diffe re nt re la tive pos itions in de oxyhe moglobin a s compa re d with oxyhe moglobin (s e e Figure 3.4). [Note : The binding of O 2 to the he me iron pulls the iron into the pla ne of the he me . Be ca use the iron is a ls o linke d to the proxima l his tidine (F8), the re is move me nt of the globin cha ins tha t a lte rs the inte rfa ce be twe e n the αβ dime rs .] a. T fo rm: The de oxy form of he moglobin is ca lle d the “T,” or ta ut

(te ns e ) form. In the T form, the two α β dime rs inte ra ct through a ne twork of ionic bonds a nd hydroge n bonds tha t cons tra in the move me nt of the polype ptide cha ins . The T conforma tion is the low-oxyge n-a ffinity form of he moglobin. b. R fo rm: The binding of O 2 to he moglobin ca us e s the rupture

of s ome of the pola r bonds be twe e n the α β dime rs , a llowing move me nt. This le a ds to a s tructure ca lle d the “R,” or re la xe d form (s e e Figure 3.4). The R conforma tion is the high-oxyge na ffinity form of he moglobin. D. Binding o f o xyg e n to myo g lo bin and he mo g lo bin Myoglobin ca n bind only one mole cule of O 2 , be ca us e it conta ins only one he me group. In contra s t, he moglobin ca n bind four O 2 mole cule s , one a t e a ch of its four he me groups . The de gre e of s a tura tion (Y) of the s e oxyge n-binding s ite s on a ll myoglobin or he moglobin mole cule s ca n va ry be twe e n ze ro (a ll s ite s a re e mpty) a nd 100% (a ll s ite s a re full), a s s hown in Figure 3.5. [Note : P uls e oxime try is a noninva s ive , indire ct me thod of me a s uring the O 2 s a tura tion of a rte ria l blood ba s e d on diffe re nce s in light a bs orption by oxyhe moglobin a nd de oxyhe moglobin.]

II. Globula r He me prote ins

29

pa rtia l pre s s ure s of oxyge n (pO 2 ) is ca lle d the oxyge n-dis s ocia tion curve . [Note : pO 2 ma y a ls o be re pre s e nte d a s P O 2 .] The curve s for myoglobin a nd he moglobin s how importa nt diffe re nce s (s e e Figure 3.5). This gra ph illus tra te s tha t myoglobin ha s a highe r oxyge n a ffinity a t a ll pO 2 va lue s tha n doe s he moglobin. The pa rtia l pre s s ure of oxyge n ne e de d to a chie ve ha lf-s a tura tion of the binding s ite s (P 50 ) is a pproxima te ly 1 mm Hg for myoglobin a nd 26 mm Hg for he moglobin. The highe r the oxyge n a ffinity (tha t is , the more tightly oxyge n binds ), the lowe r the P 50 . a. Myo g lo bin: The oxyge n-dis s ocia tion curve for myoglobin ha s

a hype rbolic s ha pe (s e e Figure 3.5). This re fle cts the fa ct tha t myoglobin re ve rs ibly binds a s ingle mole cule of oxyge n. Thus , oxyge na te d (MbO 2 ) a nd de oxyge na te d (Mb) myoglobin e xis t in a s imple e quilibrium: Mb + O 2

→ ←

The oxyg e n-dis s oc iation c urve for Hb is s te e pe s t at the oxyge n c onc e ntratio ns that oc c ur in the tis s ue s . This pe rmits o xyg e n de live ry to re s po nd to s mall c hange s in pO2 . pO2 in tis s ue s % S aturatio ratio n with O2 (Y)

1. Oxyg e n-dis s o c iatio n c urve : A plot of Y me a s ure d a t diffe re nt

Myo g lo bin

100

He mo g lo o bin n 50

0 0

MbO 2

The e quilibrium is s hifte d to the right or to the le ft a s oxyge n is a dde d to or re move d from the s ys te m. [Note : Myoglobin is de s igne d to bind oxyge n re le a s e d by he moglobin a t the low pO 2 found in mus cle . Myoglobin, in turn, re le a s e s oxyge n within the mus cle ce ll in re s pons e to oxyge n de ma nd.]

pO2 in lung s

40

80

120

Partial pre s s ure o f o xyg e n (pO2 ) (mm Hg ) P 50 = 1

P 50 = 26

Fig ure 3.5 Oxyge n-dis s ocia tion curve s for myoglobin a nd he moglobin (Hb).

b. He mo g lo bin: The oxyge n-dis s ocia tion curve for he moglobin

is s igmoida l in s ha pe (s e e Figure 3.5), indica ting tha t the s ubunits coope ra te in binding oxyge n. Coope ra tive binding of oxyge n by the four s ubunits of he moglobin me a ns tha t the binding of a n oxyge n mole cule a t one he me group incre a s e s the oxyge n a ffinity of the re ma ining he me groups in the s a me he moglobin te tra me r (Figure 3.6). This e ffe ct is re fe rre d to a s he me –he me inte ra ction (s e e be low). Although it is more difficult for the firs t oxyge n mole cule to bind to he moglobin, the s ubs e que nt binding of oxyge n occurs with high a ffinity, a s s hown by the s te e p upwa rd curve in the re gion ne a r 20–30 mm Hg (s e e Figure 3.5).

Hb O2 Hb O2

O2

Hb O2

O2

E. Allo s te ric e ffe c ts The a bility of he moglobin to re ve rs ibly bind oxyge n is a ffe cte d by the pO 2 (through he me –he me inte ra ctions a s de s cribe d a bove ), the pH of the e nvironme nt, the pa rtia l pre s s ure of ca rbon dioxide (pCO 2 ) a nd the a va ila bility of 2,3-bis phos phoglyce ra te . The s e a re colle ctive ly ca lle d a llos te ric (“othe r s ite ”) e ffe ctors , be ca us e the ir inte ra ction a t one s ite on the he moglobin mole cule a ffe cts the binding of oxyge n to he me groups a t othe r s ite s on the mole cule . [Note : The binding of oxyge n to monome ric myoglobin is not influe nce d by a llos te ric e ffe ctors .]

O2 O2 Hb O2

O2

O2

Inc re as ing

affinity

fo r

O2

O2

O2

Hb O2

O2

1. He me –he me inte rac tio ns : The s igmoida l oxyge n-dis s ocia tion

curve re fle cts s pe cific s tructura l cha nge s tha t a re initia te d a t one he me group a nd tra ns mitte d to othe r he me groups in the he moglobin te tra me r. The ne t e ffe ct is tha t the a ffinity of he moglobin for the la s t oxyge n bound is a pproxima te ly 300 time s gre a te r tha n its a ffinity for the firs t oxyge n bound.

Fig ure 3.6 He moglobin (Hb) binds s ucce s s ive mole cule s of oxyge n with incre a s ing a ffinity.

30

3. Globula r P rote ins a. Lo ading and unlo ading o xyg e n: The coope ra tive binding of

LUNGS CO2 is re le as e d fro m he mo g lo bin.

CO2

oxyge n a llows he moglobin to de live r more oxyge n to the tis s ue s in re s pons e to re la tive ly s ma ll cha nge s in the pa rtia l pre s s ure of oxyge n. This ca n be s e e n in Figure 3.5, which indica te s pO 2 in the a lve oli of the lung a nd the ca pilla rie s of the tis s ue s . For e xa mple , in the lung, the conce ntra tion of oxyge n is high, a nd he moglobin be come s virtua lly s a tura te d (or “loa de d”) with oxyge n. In contra s t, in the pe riphe ra l tis s ue s , oxyhe moglobin re le a s e s (or “unloa ds ”) much of its oxyge n for us e in the oxida tive me ta bolis m of the tis s ue s (Figure 3.7).

O2 binds to he mo g lo bin.

O2

b. S ig nific anc e o f the s ig mo idal o xyg e n-dis s o c iatio n c urve : The

NHC O O – O2

O2 Fe 2 + Fe 2 +

Fe 2 + Fe 2 +

Fe 2 + Fe 2 +

Fe 2 + Fe 2 +

NHC O O

O2

O2



Carbamino he mo g lo bin Oxyhe mo g lo bin

CO2

O2

2. Bo hr e ffe c t: The re le a s e of oxyge n from he moglobin is e nha nce d

2 CO2 binds to he mo g lo bin.

O2 is re le as e d fro m he mo g lo bin.

TIS S UES Fig ure 3.7 Tra ns port of oxyge n a nd ca rbon dioxide by he moglobin. Fe = iron.

% S aturatio n with O2 (Y)

De c re as e in pH re s ults in de c re as e d o xyg e n affinity o f he mo g lo bin and, the re fo re , a s hift to the rig ht in the o xyg e n-dis s o c iatio n c urve . pH = 7.6

100

pH = 7.2 At lo we r pH, a g re ate r pO2 is re quire d to ac hie ve any g ive n o xyg e n s aturatio n.

50

0 0

40

80

s te e p s lope of the oxyge n-dis s ocia tion curve ove r the ra nge of oxyge n conce ntra tions tha t occur be twe e n the lungs a nd the tis s ue s pe rmits he moglobin to ca rry a nd de live r oxyge n e fficie ntly from s ite s of high to s ite s of low pO 2 . A mole cule with a hype rbolic oxyge n-dis s ocia tion curve , s uch a s myoglobin, could not a chie ve the s a me de gre e of oxyge n re le a s e within this ra nge of pa rtia l pre s s ure s of oxyge n. Ins te a d, it would ha ve ma ximum a ffinity for oxyge n throughout this oxyge n pre s s ure ra nge a nd, the re fore , would de live r no oxyge n to the tis s ue s .

120

Partial pre s s ure o f o xyg e n (pO2 ) (mm Hg )

Fig ure 3.8 Effe ct of pH on the oxyge n a ffinity of he moglobin. Protons a re a llos te ric e ffe ctors of he moglobin.

whe n the pH is lowe re d or whe n the he moglobin is in the pre s e nce of a n incre a s e d pCO 2 . Both re s ult in a de cre a s e d oxyge n a ffinity of he moglobin a nd, the re fore , a s hift to the right in the oxyge n-dis s ocia tion curve (Figure 3.8), a nd both, the n, s ta bilize the T (de oxy) form. This cha nge in oxyge n binding is ca lle d the Bohr e ffe ct. Conve rs e ly, ra is ing the pH or lowe ring the conce ntra tion of CO 2 re s ults in a gre a te r a ffinity for oxyge n, a s hift to the le ft in the oxyge n-dis s ocia tion curve , a nd s ta biliza tion of the R (oxy) form. a. S o urc e o f the pro to ns that lo we r the pH: The conce ntra tion

of both H+ a nd CO 2 in the ca pilla rie s of me ta bolica lly a ctive tis s ue s is highe r tha n tha t obs e rve d in a lve ola r ca pilla rie s of the lungs , whe re CO 2 is re le a s e d into the e xpire d a ir. In the tis s ue s , CO 2 is conve rte d by ca rbonic a nhydra s e to ca rbonic a cid: CO 2 + H2 O

→ ←

H2 CO 3

which s ponta ne ous ly los e s a proton, be coming bica rbona te (the ma jor blood buffe r): H2 CO 3

→ ←

HCO 3 – + H+

The H+ produce d by this pa ir of re a ctions contribute s to the lowe ring of pH. This diffe re ntia l pH gra die nt (tha t is , lungs ha ving a highe r pH a nd tis s ue s a lowe r pH) fa vors the unloa ding of oxyge n in the pe riphe ra l tis s ue s a nd the loa ding of oxyge n in the lung. Thus , the oxyge n a ffinity of the he moglobin mole cule re s ponds to s ma ll s hifts in pH be twe e n the lungs a nd oxyge n-cons uming tis s ue s , ma king he moglobin a more e fficie nt tra ns porte r of oxyge n.

II. Globula r He me prote ins

31

b. Me c hanis m o f the Bo hr e ffe c t: The Bohr e ffe ct re fle cts the

fa ct tha t the de oxy form of he moglobin ha s a gre a te r a ffinity for protons tha n doe s oxyhe moglobin. This e ffe ct is ca us e d by ioniza ble groups s uch a s s pe cific his tidine s ide cha ins that ha ve a highe r pKa in de oxyhe moglobin tha n in oxyhe moglobin. The re fore , a n increa s e in the conce ntra tion of protons (re s ulting in a de cre a s e in pH) ca us e s the s e groups to be come protona te d (cha rge d) a nd a ble to form ionic bonds (s a lt bridge s ). The s e bonds pre fe re ntia lly s ta bilize the de oxy form of he moglobin, producing a de cre a s e in oxyge n a ffinity. [Note : He moglobin, the n, is a n importa nt blood buffe r.]

Glyc o lys is Gluc o s e

1,3-Bis phos phoglyce ra te

2,3-Bis pho s pho g lyc e rate

The Bohr e ffe ct ca n be re pre s e nte d s che ma tica lly a s : +

HbO 2 + H oxyhe moglobin

→ ←

HbH + O 2 de oxyhe moglobin

whe re a n incre a s e in protons (or a lowe r pO 2 ) s hifts the e quilibrium to the right (fa voring de oxyhe moglobin), whe re a s a n incre a s e in pO 2 (or a de cre a s e in protons ) s hifts the e quilibrium to the le ft.

O C O– HC O P HC O P H H2 O

3-P hos phoglyce ra te

P yruva te

P O 4 2–

Lac tate

3. Effe c t o f 2,3-bis pho s pho g lyc e rate o n o xyg e n affinity: 2 ,3 -

Bis phos phoglyce ra te (2,3-BP G) is a n importa nt re gula tor of the binding of oxyge n to he moglobin. It is the mos t a bunda nt orga nic phos pha te in the RBC, whe re its conce ntra tion is a pproxima te ly tha t of he moglobin. 2,3-BP G is s ynthe s ize d from a n inte rme dia te of the glycolytic pa thwa y (Figure 3.9; s e e p. 101 for a dis cus s ion of 2,3-BP G s ynthe s is in glycolys is ). a. Binding o f 2,3-BPG to de o xyhe mo g lo bin: 2,3-BP G de cre a s e s

Fig ure 3.9 S ynthe s is of 2,3-bis phos phoglyce ra te . [Note : P is a phos phoryl group, P O 3 2– .] In olde r lite ra ture , 2, 3-bis phos phoglyce ra te (2,3-BP G) ma y be re fe rre d to a s 2,3-diphos phoglyce ra te (2,3-DP G).

the O 2 a ffinity of he moglobin by binding to de oxyhe moglobin but not to oxyhe moglobin. This pre fe re ntia l binding s ta bilize s the T conforma tion of de oxyhe moglobin. The e ffe ct of binding 2,3-BP G ca n be re pre s e nte d s che ma tica lly a s : HbO 2 + 2,3-BP G oxyhe moglobin

→ ←

Hb–2,3-BP G + O 2 de oxyhe moglobin

b. Binding s ite o f 2,3-BPG: One molecule of 2,3-BPG binds to a

pocket, formed by the two β-globin chains, in the center of the de oxyhe moglobin te tra me r (Figure 3.10). This pocke t contains several positive ly cha rge d amino acids that form ionic bonds with the ne ga tive ly cha rge d phos pha te groups of 2,3-BP G. [Note : Re pla ce me nt of one of the se a mino a cids ca n re sult in he moglobin varia nts with abnormally high oxyge n affinity that may be compensated for by increased RBC production (erythrocytosis).] 2,3-BPG is expelled with oxygenation of the hemoglobin. c . S hift o f the o xyg e n-dis s o c iatio n c urve : He moglobin from

which 2,3-BP G ha s be e n re move d ha s a high a ffinity for oxyge n. Howe ve r, a s s e e n in the RBC, the pre s e nce of 2,3-BP G s ignifica ntly re duce s the a ffinity of he moglobin for oxyge n, s hifting the oxyge n-dis s ocia tion curve to the right (Figure 3.11). This re duce d a ffinity e na ble s he moglobin to re le a s e oxyge n e fficie ntly a t the pa rtia l pre s s ure s found in the tis s ue s .

A s ing le mo le c ule o f 2,3-BPG binds to a po s itive ly c harg e d c avity fo rme d by the β-c hains o f de o xyhe mo g lo bin.

α1

β2

α2

Fig ure 3.10 Binding of 2,3-bis phos phoglyce ra te (2,3-BP G) by de oxyhe moglobin.

32

3. Globula r P rote ins d. Re s po ns e o f 2,3-BP G le ve ls to c hro nic hypo xia o r ane mia: The conce ntra tion of 2,3-BP G in the RBC incre a s e s in

% S aturatio n with O2 (Y)

2,3-BPG = 0 (He mo g lo bin s trippe d o f 2,3-BPG) 100

2,3-BPG = 5 mmo l/l (No rmal blo o d)

2,3-BPG = 8 mmo l/l (Blo o d fro m individual adapte d to hig h altitude s )

re s pons e to chronic hypoxia , s uch a s tha t obs e rve d in chronic obs tructive pulmona ry dis e a s e (COP D) like e mphys e ma , or a t high a ltitude s , whe re circula ting he moglobin ma y ha ve difficulty re ce iving s ufficie nt oxyge n. Intra ce llula r le ve ls of 2,3BP G a re a ls o e le va te d in chronic a ne mia , in which fe we r tha n norma l RBCs a re a va ila ble to s upply the body’s oxyge n ne e ds . Ele va te d 2,3-BP G le ve ls lowe r the oxyge n a ffinity of he moglobin, pe rmitting gre a te r unloa ding of oxyge n in the ca pilla rie s of the tis s ue s (s e e Figure 3.11).

0 0

40

80

120

Partial pre s s ure o f o xyg e n (mm Hg )

Fig ure 3.11 Allos te ric e ffe ct of 2,3-bis phos phoglyce ra te (2,3-BP G) on the oxyge n a ffinity of he moglobin.

e . Ro le o f 2,3-BPG in trans fus e d blo o d: 2,3-BP G is e s s e ntia l for

the norma l oxyge n tra ns port function of he moglobin. Howe ve r, s toring blood in the curre ntly a va ila ble me dia re s ults in a de cre a s e in 2,3-BP G. S tore d blood dis pla ys a n a bnorma lly high oxyge n a ffinity a nd fa ils to unloa d its bound oxyge n prope rly in the tis s ue s . He moglobin de ficie nt in 2,3-BP G thus a cts a s a n oxyge n “tra p” ra the r tha n a s a n oxyge n tra ns port s ys te m. Tra ns fus e d RBC a re a ble to re s tore the ir de ple te d s upplie s of 2,3-BP G in 6–24 hours . Howe ve r, s e ve re ly ill pa tie nts ma y be compromis e d if tra ns fus e d with la rge qua ntitie s of s uch 2,3-BP G–“s trippe d” blood. [Note : The ma ximum s tora ge time for RBC ha s be e n double d (21 to 42 da ys , with me dia n time of 15 da ys ) by cha nge s in H+, phos pha te , a nd he xos e s uga r conce ntra tion a nd by the a ddition of a de nine (s e e p. 291). Although the conte nt of 2,3-BP G wa s not gre a tly improve d in the longte rm by the s e cha nge s , a de nos ine triphos pha te production wa s incre a s e d a nd improve d RBC s urviva l.] 4. Binding o f CO2 : Mos t of the CO 2 produce d in me ta bolis m is

hydra te d a nd tra ns porte d a s bica rbona te ion (s e e p. 9). Howe ve r, s ome CO 2 is ca rrie d a s ca rba ma te bound to the N-te rmina l a mino groups of he moglobin (forming ca rba minohe moglobin a s s hown in Figure 3.7), which ca n be re pre s e nte d s che ma tica lly a s follows :

O2 Co nte nt (ml/100 ml blo o d)

Hb – NH2 + CO 2 0% CO-Hb

20

→ ←

Hb – NH – COO – + H+

The binding of CO 2 s ta bilize s the T or de oxy form of he moglobin, re s ulting in a de cre a s e in its a ffinity for oxyge n (s e e p. 28) a nd a right s hift in the oxyge n-dis s ocia tion curve . In the lungs , CO 2 dis s ocia te s from the he moglobin a nd is re le a s e d in the bre a th.

50% CO-Hb 10

5. Binding o f CO: Ca rbon monoxide (CO) binds tightly (but re ve rs -

0 0

40

80

120

Partial pre s s ure o f o xyg e n (pO2 ) (mm Hg )

Fig ure 3.12 Effe ct of ca rbon monoxide (CO) on the oxyge n a ffinity of he moglobin. CO-Hb = ca rboxyhe moglobin (ca rbon monoxyhe moglobin).

ibly) to the he moglobin iron, forming ca rboxyhe moglobin. Whe n CO binds to one or more of the four he me s ite s , he moglobin s hifts to the R conforma tion, ca us ing the re ma ining he me s ite s to bind oxyge n with high a ffinity. This s hifts the oxyge n-dis s ocia tion curve to the le ft a nd cha nge s the norma l s igmoida l s ha pe towa rd a hype rbola . As a re s ult, the a ffe cte d he moglobin is una ble to re le a s e oxyge n to the tis s ue s (Figure 3.12). [Note : The a ffinity of he moglobin for CO is 220 time s gre a te r tha n for oxyge n. Cons e que ntly, e ve n minute conce ntra tions of CO in the e nvironme nt ca n produce toxic conce ntra tions of ca rboxyhe moglobin in the blood. For exa mple , incre a s e d le ve ls of CO a re found in the blood of toba cco s moke rs . CO toxicity a ppe a rs to re s ult from a

II. Globula r He me prote ins combina tion of tis s ue hypoxia a nd dire ct CO-me dia te d da ma ge a t the ce llula r le ve l.] CO pois oning is tre a te d with 100% oxyge n a t high pre s s ure (hype rba ric oxyge n the ra py), which fa cilita te s the dis s ocia tion of CO from the he moglobin. [Note : CO inhibits Comple x IV of the e le ctron tra ns port cha in (s e e p. 76).] In a ddition to O 2 , CO 2 , a nd CO, nitric oxide ga s (NO) a ls o is ca rrie d by he moglobin. NO is a pote nt va s odila tor (s e e p. 151). It ca n be ta ke n up (s a lva ge d) or re le a s e d from RBC, thus modula ting NO a va ila bility a nd influe ncing ve s s e l dia me te r. F. Mino r he mo g lo bins It is importa nt to re me mbe r tha t huma n he moglobin A (HbA) is jus t one me mbe r of a functiona lly a nd s tructura lly re la te d fa mily of prote ins , the he moglobins (Figure 3.13). Ea ch of the s e oxyge n-ca rrying prote ins is a te tra me r, compos e d of two α -globin (or α -like ) polype ptide s a nd two β-globin (or β-like ) polype ptide s . Ce rta in he moglobins , s uch a s HbF, a re norma lly s ynthe s ize d only during fe ta l de ve lopme nt, whe re a s othe rs , s uch a s HbA2 , a re s ynthe s ize d in the a dult, a lthough a t low le ve ls compa re d with HbA. HbA ca n a ls o be come modifie d by the cova le nt a ddition of a he xos e (s e e p. 34).

33

Fo rm HbA

α 2β 2

90%

HbF

α 2 γ2

1, the n ∆G o < 0

A

B

If Ke q 0

A

B

3. ∆Go o f two c o ns e c utive re ac tio ns : The ∆G o s a re a dditive in a ny

s e que nce of cons e cutive re a ctions , a s a re the ∆Gs . For e xa mple : Glucose + ATP → glucose 6-phosphate + ADP ∆G o = –4,000 cal/mol Glucos e 6-phos pha te → fructos e 6-phos pha te ∆G o = +400 ca l/mol Glucos e + ATP

→ fructos e 6-phos pha te + ADP ∆G o = –3,600 ca l/mol

4. ∆Gs o f a pathway: The a dditive prope rty of fre e e ne rgy cha nge s

C

Co upling o f a favo rable pro c e s s (– ∆G) with an unfavo rable pro c e s s (+∆G) to yie ld an o ve rall – ∆G

is ve ry importa nt in bioche mica l pa thwa ys through which s ubs tra te s mus t pa s s in a pa rticula r dire ction (for e xa mple , A → B → C → D → ...). As long a s the s um of the ∆Gs of the individua l re a ctions is ne ga tive , the pa thwa y ca n pote ntia lly proce e d a s writte n, e ve n if s ome of the individua l re a ctions of the pa thwa y ha ve a pos itive ∆G. The a ctua l ra te of the re a ctions doe s , of cours e , de pe nd on the lowe ring of a ctiva tion e ne rgie s by the e nzyme s tha t ca ta lyze the re a ctions (s e e p. 55).

IV. ADENOS INE TRIPHOS PHATE AS AN ENERGY CARRIER

−∆G +∆G

Fig ure 6.4 Me cha nica l mode l of coupling of fa vora ble a nd unfa vora ble proce s s e s . A Ge a r with we ight a tta che d s ponta ne ous ly turns in the dire ction tha t a chie ve s the B The lowe s t e ne rgy s ta te . B re ve rs e move me nt is e ne rge tica lly unfa vora ble (not s ponta nC The e ne rge tica lly fa vore ous ). C a ble move me nt ca n drive the unfa vora ble one .

Re a ctions or proce s s e s tha t ha ve a la rge pos itive ∆G, s uch a s moving ions a ga ins t a conce ntra tion gra die nt a cros s a ce ll me mbra ne , a re ma de pos s ible by coupling the e nde rgonic move me nt of ions with a s e cond, s ponta ne ous proce s s with a la rge ne ga tive ∆G s uch a s the e xe rgonic hydrolys is of a de nos ine triphos pha te (ATP ). [Note : In the a bs e nce of e nzyme s , ATP is a s ta ble mole cule be ca us e its hydrolys is ha s a high a ctiva tion e ne rgy (s e e p. 55).] Figure 6.4 s hows a me cha nica l mode l of e ne rgy coupling. The s imple s t e xa mple of e ne rgy coupling in biologic re a ctions occurs whe n the e ne rgy-re quiring a nd the e ne rgyyie lding re a ctions s ha re a common inte rme dia te . A. Co mmo n inte rme diate s Two che mica l re a ctions ha ve a common inte rme dia te whe n the y occur s e que ntia lly s o tha t the product of the firs t re a ction is a s ubs tra te for the s e cond. For e xa mple , give n the re a ctions A+ B →C + D D+ X →Y+ Z D is the common inte rme dia te a nd ca n s e rve a s a ca rrie r of che mica l e ne rgy be twe e n the two re a ctions . Ma ny couple d re a ctions us e ATP

V. Ele ctron Tra ns port Cha in to ge ne ra te a common inte rme dia te . The s e re a ctions ma y involve the tra ns fe r of a phos pha te group from ATP to a nothe r mole cule . Othe r re a ctions involve the tra ns fe r of phos pha te from a n e ne rgy-rich inte rme dia te to a de nos ine diphos pha te (ADP ), forming ATP .

73

Hig h-e ne rg y pho s phate bo nds

Ade nine

NH 2

B. Ene rg y c arrie d by ade no s ine tripho s phate ATP cons is ts of a mole cule of a de nos ine (a de nine + ribos e ) to which thre e phos pha te groups a re a tta che d (Figure 6.5). If one phos pha te is re move d, ADP is produce d. If two phos pha te s a re re move d, a de nos ine monophos pha te (AMP ) re s ults . The s ta nda rd fre e e ne rgy of hydrolys is of ATP , ∆G o , is a pproxima te ly –7.3 kca l/mol for e a ch of the two te rmina l phos pha te groups . Be ca us e of this la rge ne ga tive ∆G o , ATP is ca lle d a high-e ne rgy phos pha te compound.

V. ELECTRON TRANS PORT CHAIN

N

N

O O P O

O O P O

N

N

O O P O O

O

HO

HO

Ribo s e

Fig ure 6.5 Ade nos ine triphos pha te .

Ene rgy-rich mole cule s , s uch a s glucos e , a re me ta bolize d by a s e rie s of oxida tion re a ctions ultima te ly yie lding CO 2 a nd wa te r (Figure 6.6). The me ta bolic inte rme dia te s of the s e re a ctions dona te ele ctrons to s pe cific coe nzyme s , nicotina mide a de nine dinucle otide (NAD+) a nd fla vin a de nine dinucle otide (FAD), to form the e ne rgy-rich re duce d forms , NADH a nd FADH2 . The s e re duce d coe nzyme s ca n, in turn, e a ch dona te a pa ir of e le ctrons to a s pe cia lize d s e t of e le ctron ca rrie rs , colle ctive ly ca lle d the e le ctron tra ns port cha in (ETC), de s cribe d in this s e ction. As e le ctrons a re pa s s e d down the ETC, the y los e much of the ir fre e e ne rgy. This e ne rgy is us e d to move protons a cros s the inne r mitochondria l me mbra ne , cre a ting a proton gra die nt tha t drive s the production of ATP from ADP a nd inorga nic phos pha te (P i), de s cribe d on p. 77. The coupling of e le ctron tra ns port with ATP s ynthe s is is ca lle d oxida tive phos phoryla tion, ofte n de note d a s OXP HOS . It proce e ds continuous ly in a ll tis s ue s tha t conta in mitochondria . [Note : The re ma inde r of the fre e e ne rgy not tra ppe d a s ATP is us e d to drive a ncilla ry re a ctions s uch a s ca lcium tra ns port into mitochondria (s e e p. 133) a nd to ge ne ra te he a t.]

Me tabo lis m Carbo hydrate s Fatty ac ids Amino ac ids +

NAD FAD

NADH + H+ FADH2 CO2 + H2 O

A. The e le c tro n trans po rt c hain o f the mito c ho ndrio n The ETC (e xce pt for cytochrome c; s e e p. 75) is loca te d in the inne r mitochondria l me mbra ne a nd is the fina l common pa thwa y by which e le ctrons de rive d from diffe re nt fue ls of the body flow to oxyge n (O 2 ).

O2 NADH + H+ FADH2

ADP + P i

NAD+

1. Me mbrane s o f the mito c ho ndrio n: The mitochondrion conta ins

a n oute r a nd a n inne r me mbra ne s e pa ra te d by the inte rme mbra ne s pa ce . Although the oute r me mbra ne conta ins s pe cia l cha nne ls (forme d by the prote in porin), ma king it fre e ly pe rme a ble to mos t ions a nd s ma ll mole cule s , the inne r me mbra ne is a s pe cia lize d s tructure tha t is impe rme a ble to mos t s ma ll ions , including protons a nd s ma ll mole cule s s uch a s ATP , ADP , pyruva te , a nd othe r me ta bolite s importa nt to mitochondria l function (Figure 6.7). S pe cia lize d ca rrie rs or tra ns port s ys te ms a re re quire d to move ions or mole cule s a cros s this me mbra ne . The inne r mitochondria l me mbra ne is unus ua lly rich in prote in, ove r ha lf of which is dire ctly involve d in oxida tive phos phoryla tion. It a ls o is highly convolute d. The convolutions , ca lle d cris ta e , s e rve to gre a tly incre a s e the s urfa ce a re a of the inne r me mbra ne .

ATP

FAD H2 O

Oxidative pho s pho rylatio n Fig ure 6.6 The me ta bolic bre a kdown of e ne rgyyie lding mole cule s . NAD(H) = nicotina mide a de nine dinucle otide ; FAD(H2 )= fla vin a de nine dinucle otide ; ADP = a de nos ine diphos pha te ; ATP = a de nos ine triphos pha te ; P i = inorga nic phos pha te .

74

6. Bioe ne rge tics a nd Oxida tive P hos phoryla tion 2. Matrix o f the mito c ho ndrio n: This ge l-like s olution in the inte rior INNER MEMBRANE Impe rme able to mo s t s mall io ns , s mall and larg e mo le c ule s

CELL

of mitochondria is a ls o rich in prote in. The s e mole cule s include the e nzyme s re s pons ible for the oxida tion of pyruva te , a mino a cids , a nd fa tty a cids (by β-oxida tion) a s we ll a s thos e of the trica rboxylic a cid (TCA) cycle . The s ynthe s is of glucos e , ure a , a nd he me occurs pa rtia lly in the ma trix of mitochondria . In a ddition, the ma trix conta ins NAD+ a nd FAD (the oxidize d forms of the two coe nzyme s tha t a re re quired a s hydroge n a cce ptors ), a nd ADP a nd P i, which a re us e d to produce ATP . [Note : The ma trix a ls o conta ins mitochondria l DNA (mtDNA) a nd RNA (mtRNA) a nd ribos ome s .] B. Org anizatio n o f the e le c tro n trans po rt c hain

Oute r me mbrane Inte rme mbrane s pac e

Cris tae NAD+

FMN Co Q

b

ADP

c

a

ATP

a3

The inne r mitochondria l me mbra ne conta ins five s e pa ra te prote in comple xe s , ca lle d Comple xe s I, II, III, IV, a nd V. Comple xe s I–IV e a ch conta in pa rt of the ETC (Figure 6.8). The s e comple xe s a cce pt or dona te e le ctrons to the re la tive ly mobile e le ctron ca rrie rs , coe nzyme Q a nd cytochrome c. Ea ch ca rrie r in the ETC ca n re ce ive e le ctrons from a n e le ctron donor a nd ca n s ubs e que ntly dona te e le ctrons to the ne xt a cce ptor in the cha in. The e lectrons ultima te ly combine with O 2 a nd protons to form wa te r. This re quire me nt for O 2 ma ke s the e le ctron tra ns port proce s s the re s pira tory cha in , which a ccounts for the gre a te s t portion of the body’s us e of O 2 . Comple x V is de s cribe d on p. 78. C. Re ac tio ns o f the e le c tro n trans po rt c hain

An e le c tro n trans po rt as s e mbly

Ade no s ine tripho s phate (ATP) s ynthe s izing s truc ture

MATRIX

• TCA c yc le e nzyme s • Fatty ac id o xidatio n e nzyme s • mtDNA, mtRNA • Mito c ho ndrial ribo s o me s Fig ure 6.7 S tructure of a mitochondrion s howing s che ma tic re pre s e nta tion of the e le ctron tra ns port cha in a nd the ATP s ynthe s izing s tructure on the inne r me mbra ne . [Note : In contra s t to the inne r me mbra ne , the oute r me mbra ne is highly pe rme a ble , a nd the milie u of the inte rme mbra ne s pa ce is like tha t of the cytos ol.] mtDNA = mitochondria l DNA; mtRNA = mitochondria l RNA; TCA = trica rboxylic a cid.

With the e xce ption of coe nzyme Q, which is a lipid-s oluble quinone , a ll me mbe rs of this cha in a re prote ins . The s e ma y function as e nzyme s a s is the ca s e with the fla vin-conta ining de hydroge na s e s, ma y conta in iron a s pa rt of a n iron-s ulfur ce nte r, ma y conta in iron a s pa rt of the porphyrin pros the tic group of he me a s in the cytochromes , or ma y conta in coppe r a s doe s the cytochrome a + a 3 comple x. 1. Fo rmatio n o f NADH: NAD+ is re duce d to NADH by de hydroge na s e s tha t re move two hydroge n a toms from the ir s ubs tra te . (For e xa mple s of the s e re a ctions , s e e the dis cus s ion of the de hydroge na s e s of the TCA cycle , p. 112.) Both e le ctrons but only one proton (tha t is , a hydride ion [:H– ]) a re tra ns fe rre d to the NAD+, forming NADH plus a fre e proton. 2. NADH de hydro g e nas e : The fre e proton plus the hydride ion ca r-

rie d by NADH a re tra nsfe rre d to NADH de hydroge na s e , a prote in comple x (Comple x I) e mbe dde d in the inne r mitochondria l me mbra ne . Comple x I ha s a tightly bound mole cule of fla vin mononucle otide (FMN), a coe nzyme s tructura lly re la te d to FAD (s e e Figure 28.15, p. 380) tha t a cce pts the two hydroge n a toms (2e – + 2H+), be coming FMNH2 . NADH de hydroge na s e a ls o conta ins pe ptide s ubunits with iron-s ulfur ce nte rs (Figure 6.9). At Comple x I, e le ctrons move from NADH to FMN to the iron of the iron-s ulfur ce nte rs a nd the n to coe nzyme Q. As e le ctrons flow, the y lose e ne rgy. This e ne rgy is us e d to pump protons a cros s the inne r mitochondria l me mbra ne , from the ma trix to the inte rme mbra ne s pa ce . 3. S uc c inate de hydro g e nas e : At Comple x II, e le ctrons from the s uc-

cina te de hydroge na s e –ca ta lyze d oxida tion of s uccina te to fuma ra te move from the coe nzyme , FADH2 , to a n iron-s ulfur prote in,

V. Ele ctron Tra ns port Cha in

75

Cyto c hro me c

Inte rme mbrane s pac e Inne r mito c ho ndrial me mbrane NADH + H+

NAD+

NADH d e h yd ro g e n a s e FMN Fe -S

Co mple x I S uc c inate

Fumarate

S u c c in a te d e h yd ro g e n a s e FAD Fe -S

Co Q/Co QH2

Cyto c hro me b

Cu A

Fe -S

Cyto c hro me a

Cyto c hro me c 1

Cu B -Cyto c hro me a 3

1 2

Co mple x III (Cyto c hro me bc 1 )

Co mple x IV (Cyto c hro me a + a 3 )

H2 O

O2

Co mple x II Fig ure 6.8 Ele ctron tra ns port cha in. The flow of e le ctrons is s hown by the ma ge nta a rrows . NAD(H) = nicotina mide a de nine dinucle otide ; FMN = fla vin mononucle otide ; FAD = fla vin a de nine dinucle otide ; Fe -S = iron-s ulfur ce nte r; CoQ = coe nzyme Q.

a nd the n to coe nzyme Q. [Note : No e ne rgy is los t in this proce s s , a nd, the re fore , no protons a re pumpe d a t Comple x II.] 4. Co e nzyme Q: Coe nzyme Q (CoQ) is a quinone de riva tive with

a long, hydrophobic is opre noid ta il. It is a ls o ca lle d ubiquinone be ca us e it is ubiquitous in biologic s ys te ms . CoQ is a mobile e le ctron ca rrie r a nd ca n a cce pt hydroge n a toms from NADH de hydroge na s e (Comple x I), from s uccina te de hydroge na s e (Comple x II), a nd from othe r mitochondria l de hydroge na s e s : glyce rophos pha te de hydroge na s e (s e e p. 79) a nd a cyl CoA de hydroge na s e (s e e p. 192). CoQ tra ns fe rs e le ctrons to Comple x III (cytochrome bc 1 ). CoQ, the n, links the fla voprote in de hydroge na s e s to the cytochrome s . NADH de hydroge na s e prote in

5. Cyto c hro me s : The re ma ining me mbe rs of the ETC a re cyto-

chrome prote ins . Ea ch conta ins a he me group (a porphyrin ring plus iron). Unlike the heme groups of hemoglobin, the cytochrome iron is re versibly converted from its fe rric (Fe 3+) to its ferrous (Fe 2+) form a s a norma l part of its function as an a cceptor a nd donor of e lectrons. Electrons a re pas se d a long the cha in from cytochrome bc 1 (Comple x III), to cytochrome c, a nd the n to cytochrome s a + a 3 (Comple x IV; s e e Figure 6.8). As e le ctrons flow, protons a re pumped across the inne r mitochondria l membrane at Complexe s III a nd IV. [Note: Cytochrome c is located in the inte rmembra ne space, loosely associa ted with the outer face of the inner me mbrane . As seen with CoQ, cytochrome c is a mobile ca rrier of electrons.] 6. Cyto c hro me a + a 3 : This cytochrome comple x (Comple x IV) is the

only e le ctron ca rrie r in which the he me iron ha s a n a va ila ble coordina tion s ite tha t ca n re a ct dire ctly with O 2 a nd s o a ls o is ca lle d cytochrome oxida s e. At Comple x IV, the tra ns porte d e le ctrons , O 2 , a nd fre e protons a re brought toge the r, a nd O 2 is re duce d to wa te r (s e e Figure 6.8). [Note : Four e le ctrons a re re quire d to re duce one mole cule of O 2 to two mole cule s of wa te r.] Cytochrome oxida s e

S Cys

S

S

Fe S

Fe

Fe S

Cys

S

S Fe S

Cys

Cys

Fe 4 S 4

Fig ure 6.9 Iron-s ulfur (Fe -S ) ce nte r of Comple x I. [Note : Comple xe s II a nd III a ls o conta in iron-s ulfur ce nte rs .] NADH = nicotina mide a de nine dinucle otide ;Cys = cys te ine .

76

6. Bioe ne rge tics a nd Oxida tive P hos phoryla tion

Blo c king e le c tro n trans fe r by any o ne o f the s e inhibito rs s to ps e le c tro n flo w fro m s ubs trate to o xyg e n be c aus e the re ac tio ns o f the e le c tro n trans po rt c hain are tig htly c o uple d like me s he d g e ars .

S ubs trate (re duc e d)

e– NAD+

e– FMN

Am y t a l Ro t e n o n e

e–

conta ins coppe r (Cu) a toms tha t a re re quire d for this complica te d re a ction to occur. Ele ctrons move from Cu A to cytochrome a to cytochrome a 3 (in a s s ocia tion with Cu B) to O 2 . 7. S ite -s pe c ific inhibito rs : S ite -s pe cific inhibitors of e le ctron tra ns -

port ha ve be e n ide ntifie d a nd a re illus tra te d in Figure 6.10. The s e compounds pre ve nt the pa s s a ge of e le ctrons by binding to a compone nt of the cha in, blocking the oxida tion-re duction re a ction. The re fore , a ll e le ctron ca rrie rs be fore the block a re fully re duce d, whe re a s thos e loca te d a fte r the block a re oxidize d. [Note : Inhibition of e le ctron tra ns port inhibits ATP s ynthe s is be ca us e the s e proce s s e s a re tightly couple d (s e e p. 77).] Incomple te re duction of oxyge n to wa te r produce s re a ctive oxyge n s pe cie s (ROS ), s uch a s s upe roxide (O 2 –•), hydroge n pe roxide (H2 O 2 ), a nd hydroxyl ra dica ls (OH•). ROS da ma ge DNA a nd prote ins a nd ca us e lipid pe roxida tion. Enzyme s s uch a s s upe roxide dis muta s e (S OD), ca ta la s e , a nd gluta thione pe roxida s e a re ce llula r de fe ns e s a ga ins t ROS .

Co Q

D. Re le as e o f fre e e ne rg y during e le c tro n trans po rt

e– Cyto bc 1

An t im y c in A

e– Cyto c

e– Cyto a + a 3 CN– CO H2S Na N3

e– O2

The fre e e ne rgy re le a s e d a s e le ctrons a re tra ns fe rre d a long the ETC from a n e le ctron donor (re ducing a ge nt or re ducta nt) to a n e le ctron a cce ptor (oxidizing a ge nt or oxida nt) is us e d to pump protons a t Comple xe s I, III, a nd IV. [Note : The e le ctrons ca n be tra ns fe rre d a s hydride ions (:H– ) to NAD+; a s hydroge n a toms (•H) to FMN, CoQ, a nd FAD; or a s e le ctrons (e – ) to cytochrome s .] 1. Re do x pairs : Oxida tion (los s of e le ctrons ) of one s ubs ta nce is

a lwa ys a ccompa nie d by re duction (ga in of e le ctrons ) of a s e cond. For e xa mple , Figure 6.11 s hows the oxida tion of NADH to NAD+ by NADH de hydroge na s e a t Comple x I, a ccompa nie d by the re duction of FMN, the pros the tic group, to FMNH2 . S uch oxida tion-re duction re a ctions ca n be writte n a s the s um of two s e pa ra te ha lf-re a ctions , one a n oxida tion a nd the othe r a re duction (s e e Figure 6.11). NAD+ a nd NADH form a re dox pa ir, a s do FMN a nd FMNH2 . Re dox pa irs diffe r in the ir te nde ncy to los e e le ctrons . This te nde ncy is a cha ra cte ris tic of a pa rticula r re dox pa ir a nd ca n be qua ntita tive ly s pe cifie d by a cons ta nt, E o (the s ta nda rd re duction pote ntia l), with units in volts . 2. S tandard re duc tio n po te ntial: The E o of va rious re dox pa irs ca n

Fig ure 6.10 S ite -s pe cific inhibitors of e le ctron tra ns port s hown us ing a me cha nica l mode l for the coupling of oxida tionre duction re a ctions . [Note : Figure illus tra te s norma l dire ction of e le ctron flow.] CN– = cya nide ; CO = ca rbon monoxide ; H2 S = hydroge n s ulfide ; Na N3 = s odium a zide ; FMN = fla vin mononucle otide ; FAD = fla vin a de nine dinucle otide ; CoQ = coe nzyme Q; Cyto = cytochrome .

be orde re d from the mos t ne ga tive E o to the mos t pos itive . The more ne ga tive the E o of a re dox pa ir, the gre a te r the te nde ncy of the re ducta nt me mbe r of tha t pa ir to los e e le ctrons . The more pos itive the E o , the gre a te r the te nde ncy of the oxida nt me mbe r of tha t pa ir to a cce pt e le ctrons . The re fore , e le ctrons flow from the pa ir with the more ne ga tive E o to tha t with the more pos itive E o . The E o va lue s for s ome me mbe rs of the ETC a re s hown in Figure 6.12. [Note : The compone nts of the cha in a re a rra nge d in orde r of incre a s ingly pos itive E o va lue s .] 3. Re latio ns hip o f ∆Go to ∆Eο : The ∆G o is re la te d dire ctly to the

ma gnitude of the cha nge in E o : ∆G o = – n F ∆E o

VI. Oxida tive P hos phoryla tion whe re

n = numbe r of e le ctrons tra ns fe rre d (1 for a cytochrome , 2 for NADH, FADH2 , a nd coe nzyme Q) F = Fa ra da y cons ta nt (23.1 kca l/volt mol) ∆E o = E o of the e le ctron-a cce pting pa ir minus the E o of the e le ctron-dona ting pa ir ∆G o = cha nge in the s ta nda rd fre e e ne rgy

77 Ove rall o xidatio n-re duc tio n re ac tio n NADH + H+

NAD+

FMN

FMNH2

Co mpo ne nt re do x re ac tio ns o

o

4. ∆G o f ATP: The ∆G for the phos phoryla tion of ADP to ATP is

+7.3 kca l/mol. The tra ns port of a pa ir of e le ctrons from NADH to O 2 through the ETC re le a s e s 52.58 kca l. The re fore , more tha n s ufficie nt e ne rgy is a va ila ble to produce 3 ATP from 3 ADP a nd 3 P i (3 x 7.3 = 21.9 kca l/mol), s ome time s e xpre s s e d a s a P :O ra tio (ATP ma de pe r O a tom re duce d) of 3:1. The re ma ining ca lorie s a re us e d for a ncilla ry re a ctions or re le a s e d a s he a t. [Note : The P :O for FADH2 is 2:1 be ca us e Comple x I is bypa s s e d.]

VI. PHOS PHORYLATION OF ADP TO ATP The tra ns fe r of e le ctrons down the ETC is e ne rge tica lly fa vore d be ca us e NADH is a s trong e le ctron donor a nd O 2 is a n a vid e le ctron a cce ptor. Howe ve r, the flow of e le ctrons doe s not directly res ult in ATP s ynthe s is .

NADH + H+

NAD++ 2e – + 2H+ Re do x pair Eo = – 0.32 vo lt

FMN + 2e – + 2H+

FMNH2 Re do x pair Eo = – 0.22 vo lt

Fig ure 6.11 Oxida tion of NADH by FMN, s e pa ra te d into two compone nt ha lf-re a ctions . NAD(H) = nicotina mide a de nine dinucle otide ; FMN(H2 ) = fla vin mononucle otide .

A. Che mio s mo tic hypo the s is The che mios motic hypothe s is (a ls o known a s the Mitche ll hypothe s is ) e xpla ins how the fre e e ne rgy ge ne ra te d by the tra nsport of e le ctrons by the ETC is us e d to produce ATP from ADP + P i.

Co mpo unds with a larg e ne g ative Eo (lo c ate d at to p o f the table ) are s tro ng re duc ing ag e nts , o r re duc tants (that is , the y have a s tro ng te nde nc y to lo s e e le c tro ns ).

1. Pro to n pump: Ele ctron tra ns port is couple d to the phos phoryla -

tion of ADP by the pumping of protons a cros s the inne r mitochondria l me mbra ne , from the ma trix to the inte rme mbra ne s pa ce , a t Comple xe s I, III, a nd IV. This proce s s cre a te s a n e le ctrica l gra die nt (with more positive cha rge s on the outs ide of the me mbra ne tha n on the ins ide ) a nd a pH gra die nt (the outs ide of the membra ne is a t a lowe r pH tha n the ins ide ) a s s hown in Figure 6.13. The e ne rgy ge ne ra te d by this proton gra die nt is s ufficie nt to drive ATP s ynthe s is . Thus , the proton gra die nt s e rve s a s the common inte rme dia te tha t couple s oxida tion to phos phoryla tion. 2. ATP s ynthas e : The multis ubunit e nzyme ATP s yntha s e (Comple x

V; s e e Figure 6.14) s ynthe s ize s ATP us ing the e ne rgy of the proton gra die nt. It conta ins a doma in (F o ) tha t s pa ns the inne r mitochondria l me mbra ne , a nd a n e xtra me mbra nous doma in (F 1 ) tha t a ppe a rs a s a s phe re tha t protrude s into the mitochondria l ma trix (s e e Figure 6.13). The che mios motic hypothe s is propos e s tha t a fte r protons ha ve be e n pumpe d to the cytos olic s ide of the inne r mitochondria l me mbra ne , the y re e nte r the ma trix by pa s s ing through a proton cha nne l in the F o doma in, driving rota tion of the c ring of F o a nd, a t the s a me time , dis s ipa ting the pH a nd e le ctrica l gra die nts . F o rota tion ca us e s conforma tiona l cha nge s in the β s ubunits of the F 1 doma in tha t a llow the m to bind ADP + P i, phos phoryla te ADP to ATP , a nd re le a s e ATP . [Note : ATP s yntha s e is a ls o ca lle d F 1 /F o -ATP a s e be ca us e the is ola te d e nzyme ca n ca ta lyze the hydrolys is of ATP to ADP a nd P i.]

Re do x pair

Eo

NAD+/NADH

-0.32

FMN/FMNH2 Cytochrome c Fe 3 +/Fe 2 + 1/2 O 2 /H2 O

-0.22 +0.22 +0.82

Co mpo unds at the bo tto m o f the table are s tro ng o xidizing ag e nts , o r o xidants (that is , the y ac c e pt e le c tro ns ).

Fig ure 6.12 S ta nda rd re duction pote ntia ls (E o ) of s ome re a ctions . NAD(H) = nicotina mide a de nine dinucle otide ; FMN(H2 ) = fla vin mononucle otide .

78

6. Bioe ne rge tics a nd Oxida tive P hos phoryla tion

MITOCHONDRION Inne r me mbrane

ADP + P i

Oute r me mbrane

ATP Co mple x V (F1 do main)

Inte rme mbrane s pac e

Matrix

NAD+

1/ 2

NADH MITOCHONDRIAL MATRIX e–

O2

H2 O ATP/ADP antipo rte r

e–

e– Co mple x I INTERMEMBRANE S P ACE

Ele c tro n flo w Co mple x III

+ H

c ytc e – Co mple x IV

+ H

Co mple x V (F0 do main)

+ H

H

+

ADP ATP

Fig ure 6.13 Ele ctron tra ns port cha in s hown in a s s ocia tion with the tra ns port of protons (H+). A tota l of te n H+ a re pumpe d for e a ch nicotina mide a de nine dinucle otide (NADH) oxidize d. [Note : H+ a re not pumpe d a t Comple x II.] a. Co upling in o xidative pho s pho rylatio n: In norma l mitochon-

β δ

α β

α

ADP + P i ATP α β

F 1 in the mitochondria l matrix conta ins the ca ta lytic a ctivity.

b2

b. Olig o myc in: This drug binds to the F o (he nce the le tte r “o”)

H+

a

dria , ATP s ynthe s is is couple d to e le ctron tra ns port through the proton gra die nt. Incre a s ing (or de cre a s ing) one proce s s ha s the s a me e ffe ct on the othe r. For e xa mple , hydrolys is of ATP to ADP a nd P i in e ne rgy-re quiring re a ctions incre a s e s the a va ila bility of s ubs tra te s for ATP s yntha s e a nd, thus , incre a s e s proton flow through the e nzyme . Ele ctron tra ns port a nd proton pumping by the ETC incre a s e to ma inta in the proton gra die nt. [Note : Incre a s e d oxida tion of NADH a t Comple x I a nd, cons e que ntly, a n incre a s e in NADH-producing pa thwa ys of me ta bolis m, s uch a s the TCA cycle , re s ults .]

γ

C

C

C

ε

C

C

F o in the inne r mitochondria l me mbra ne conta ins the proton (H+) pore .

H+

Fig ure 6.14 ATP s yntha s e (F 1 F o −ATP a s e ). [Note : The rota tion of the ring of c s ubunits in the F o doma in re s ults in conforma tiona l cha nge s in the β s ubunits of the F 1 doma in tha t a llow phos phoryla tion of a de nos ine diphos pha te (ADP ) to a de nos ine triphos pha te (ATP ). P i = inorga nic phos pha te .

doma in of ATP s yntha s e , clos ing the proton cha nne l a nd pre ve nting re e ntry of protons into the ma trix, the re by pre ve nting phos phoryla tion of ADP to ATP . Be ca us e the pH a nd e le ctrica l gra die nts ca nnot be dis s ipa te d in the pre s e nce of this drug, e le ctron tra ns port s tops be ca us e of the difficulty of pumping a ny more protons a ga ins t the s te e p gra die nts . This de pe nde ncy of ce llula r re s pira tion on the a bility to phos phoryla te ADP to ATP is known a s re s pira tory control a nd is the cons e que nce of the tight coupling of the s e proce s s e s . c . Unc o upling pro te ins : Uncoupling prote ins (UCPs ) occur in the

inne r mitochondria l membra ne of ma mma ls , including huma ns . The s e prote ins form cha nne ls tha t a llow protons to re e nte r the mitochondria l ma trix without e ne rgy be ing ca pture d a s ATP (Figure 6.15). The e nergy is re le a s e d a s he a t, a nd the proce s s is ca lle d nons hive ring the rmoge ne s is . UCP 1, a ls o ca lle d the r-

VI. Oxida tive P hos phoryla tion

79

moge nin, is re s pons ible for he a t production in the brown a dipocyte s of ma mma ls . In brown fa t, unlike the more a bunda nt white fa t, a lmos t 90% of its re s pira tory e ne rgy is us e d for the rmoge ne s is in re s pons e to cold in the ne ona te a nd during a rous a l in hibe rna ting a nima ls . Howe ve r, huma ns a ppe a r to ha ve fe w conce ntra te d de pos its of brown fa t (e xce pt in the ne wborn), a nd UCP 1 doe s not a ppe a r to pla y a ma jor role in e ne rgy ba la nce . [Note : Uncoupling prote ins UCP 2–UCP 5 ha ve be e n found in othe r tis s ue s , but the ir full s ignifica nce re ma ins uncle a r.]

Unc o upling pro te ins c re ate a c hanne l, allo wing pro to ns (H+1 ) to re e nte r the mito c ho ndrial matrix witho ut c apturing any e ne rg y as ATP.

B. Me mbrane trans po rt s ys te ms The inne r mitochondria l me mbra ne is impe rme a ble to mos t cha rge d or hydrophilic s ubs ta nce s . Howe ve r, it conta ins nume rous tra ns port prote ins tha t pe rmit pa s s a ge of s pe cific mole cule s from the cytos ol (or more corre ctly, the inte rme mbra ne s pa ce ) to the mitochondria l ma trix. 1. ATP and ADP trans po rt: The inne r me mbra ne re quire s s pe cia lize d

ca rrie rs to tra ns port ADP a nd P i from the cytos ol (whe re ATP is hydrolyze d to ADP in ma ny e ne rgy-re quiring re a ctions ) into mitochondria , whe re ATP ca n be re s ynthe s ize d. An a de nine nucle otide a ntiporte r imports one ADP from the cytos ol into the ma trix, while e xporting one ATP from the ma trix into the cytos ol (s e e Figure 6.13). A tra ns porte r move s P i from the cytos ol into mitochondria . 2. Trans po rt o f re duc ing e quivale nts : The inne r mitochondria l

me mbra ne la cks a n NADH tra ns porte r, a nd NADH produce d in the cytos ol (for e xa mple , in glycolys is ; s e e p. 101) ca nnot dire ctly e nte r the mitochondria l ma trix. Howe ve r, two e le ctrons (re ducing e quiva le nts ) of NADH a re tra ns porte d from the cytos ol into the ma trix us ing s ubs tra te s huttle s . In the glyce rophos pha te s huttle (Figure 6.16A), two e le ctrons a re tra ns fe rre d from NADH to dihydroxya ce tone phos pha te by cytos olic glyce rophos pha te de hydroge na s e . The glyce rol 3-phos pha te produce d is oxidize d by the mitochondria l is ozyme a s FAD is re duce d to FADH2 . CoQ of the ETC oxidize s the FADH2 . The glyce rophos pha te s huttle , the re fore , re s ults in the s ynthe s is of two ATP s for e a ch cytos olic NADH oxidize d. This contra s ts with the ma la te -a s pa rta te s huttle (Figure 6.16B), which produce s NADH (ra the r tha n FADH2 ) in the mitochondria l ma trix a nd, the re fore , yie lds thre e ATP s for e a ch cytos olic NADH oxidize d by ma la te de hydroge na s e a s oxa loa ce ta te is re duce d to ma la te . A tra ns port prote in move s ma la te into the ma trix.

H

H +

+

H

d. S ynthe tic unc o uple rs : Ele ctron tra ns port a nd phos phoryla tion

ca n a ls o be uncouple d by compounds tha t pick up protons in the inte rme mbra ne s pa ce a nd re le a s e the m in the ma trix, dis s ipa ting the gra die nt. The cla s s ic e xa mple is 2,4-dinitrophe nol, a lipophilic proton ca rrie r tha t re a dily diffus e s through the mitochondria l me mbra ne . This uncouple r ca us e s e le ctron tra ns port to proce e d a t a ra pid ra te without e s ta blis hing a proton gra die nt, much a s do the UCP s (s e e Figure 6.15). Aga in, e ne rgy is re le a s e d a s he a t ra the r tha n be ing us e d to s ynthe s ize ATP . [Note : In high dos e s , a s pirin a nd othe r s a licyla te s uncouple oxida tive phos phoryla tion. This e xpla ins the fe ve r tha t a ccompa nie s toxic ove rdos e s of the s e drugs .]

+

+

Unc o upling pro te in

H

+

H

ATP s yn th a s e

O2

+

H H2 O

+ ADP

H

e–

ATP

MITOCHONDRIAL MATRIX

Fig ure 6.15 Tra ns port of protons a cros s the mitochondria l me mbra ne by a n uncoupling prote in. ADP = a de nos ine diphos pha te ; ATP = a de nos ine triphos pha te .

80

6. Bioe ne rge tics a nd Oxida tive P hos phoryla tion C. Inhe rite d de fe c ts in o xidative pho s pho rylatio n

A NADH + H+

NAD+

CH2 OH

CH2 OH

C O

Cytos olic CH2 OP O 3 glyce rophos pha te de hydroge na s e

DHAP

HO C H CH2 OP O 3 Glyc e ro l 3-pho s phate

CYTOS OL

Co Q o f the e le c tro n trans po rt c hain FADH2

CH2 OH

C O

FAD CH2 OH

Mitochondria l glyce rophos pha te de hydroge na s e

CH2 OP O 3 DHAP

HO C H CH2 OP O 3 Glyc e ro l 3-pho s phate

INNER MITOCHONDRIAL MEMBRANE

B Oxalo ac e tate NADH + H+

Cytos olic ma la te de hydroge na s e

Glutamate

The proce s s of a poptos is , or progra mme d ce ll de a th, ma y be initia te d through the intrins ic (mitochondria l-me dia te d) pa thwa y by the forma tion of pore s in the oute r mitochondria l me mbra ne . The s e pore s a llow cytochrome c to le a ve the inte rme mbra ne s pa ce a nd e nte r the cytos ol. The re , cytochrome c, in a s s ocia tion with proa poptotic fa ctors , a ctiva te s a fa mily of prote olytic e nzyme s (the ca s pa s e s ), ca us ing cle a va ge of ke y prote ins a nd re s ulting in the morphologic a nd bioche mica l cha nge s cha ra cte ris tic of a poptos is .

VII. CHAPTER S UMMARY

Malate As partate α-Ke to g lutarate CYTOS OL

As partate α-Ke to g lutarate

Malate NAD+

Aminotra ns fe ra s e

NADH + H+ Oxalo ac e tate

D. Mito c ho ndria and apo pto s is

Aminotra ns fe ra s e

NAD+

Mitochondria l ma la te de hydroge na s e

Thirte e n of the a pproxima te ly 90 polype ptide s re quire d for oxida tive phos phoryla tion a re code d for by mtDNA a nd s ynthe s ize d in mitochondria , whe re a s the re ma ining prote ins a re code d for by nucle a r DNA, s ynthe s ize d in the cytos ol, a nd tra ns porte d into mitochondria pos ttra ns la tiona lly. De fe cts in oxida tive phos phoryla tion a re more like ly a result of alterations in mtDNA, which has a muta tion rate a bout 10 times greater than tha t of nuclear DNA. Tissue s with the greatest ATP requirement (for exa mple, ce ntra l ne rvous sys tem, s keletal a nd heart muscle , and liver) a re most a ffected by defects in oxidative phosphorylation. Mutations in mtDNA a re responsible for se vera l diseases, including some cases of mitochondrial myopathies, and Lebe r hereditary optic ne uropathy, a disease in which bilate ral loss of ce ntral vision occurs as a re sult of neuroretinal degene ration, including damage to the optic nerve . [Note : mtDNA is materna lly inhe rited be ca us e mitochondria from the sperm cell do not enter the fe rtilized egg.]

Glutamate

Co mple x I o f the e le c tro n trans po rt c hain MITOCHONDRIAL MATRIX

Fig ure 6.16 S ubs tra te s huttle s for the tra ns port of e le ctrons a cros s the inne r mitochondria l me mbra ne . A. Glyce rophos pha te s huttle . B. Ma la te -a s pa rta te s huttle . DHAP = dihydroxya ce tone phos pha te ; NAD(H) = nicotina mide a de nine dinucle otide ; FAD(H2 ) = fla vin a de nine dinucle otide ; CoQ = coe nzyme Q.

The cha nge in fre e e ne rg y (∆G) occurring during a re a ction pre dicts the dire ction in which tha t re a ction will s ponta ne ous ly proce e d. If ∆G is ne g ative (tha t is , the product ha s a lowe r fre e e ne rgy tha n the s ubs tra te ), the re ac tio n g o e s s po ntane o us ly . If ∆G is po s itive , the re a ction do e s no t g o s po ntane o us ly . If ∆G = 0 , the re a ctions a re in e quilibrium . The ∆G of the forwa rd re a ction is e qua l in ma gnitude but oppos ite in s ign to tha t of the ba ck re a ction. The ∆Gs a re additive in a ny s e que nce of cons e cutive re a ctions , a s a re the s ta nda rd fre e e ne rgy cha nge s (∆Go s ). The re fore , re a ctions or proce s s e s tha t ha ve a la rge , pos itive ∆G a re ma de pos s ible by c o upling with thos e tha t ha ve a la rge , ne ga tive ∆G s uch a s hydrolys is of ade no s ine tripho s phate (ATP). The re duce d coe nzyme s nic o tinamide ade nine dinuc le o tide (NADH) a nd flavin ade nine dinuc le o tide ( FADH2 ) e a ch dona te a pa ir of e le ctrons to a s pe cia lize d s e t of e le ctron ca rrie rs , cons is ting of flavin mo no nuc le o tide (FMN), iro n-s ulfur c e nte rs , c o e nzyme Q, a nd a s e rie s of c yto c hro me s , colle ctive ly ca lle d the e le c tro n trans po rt c hain . This pa thwa y is pre s e nt in the inne r mito c ho ndrial me mbrane (impe rme a ble to mos t s ubs ta nce s ) a nd is the fina l common pa thwa y by which e le ctrons de rive d from diffe re nt fue ls of the body flow to O 2 , re ducing it to wa te r. The te rmina l cytochrome , cytochrome oxida s e , is the only cytochrome a ble to bind O 2 . Ele c tro n trans po rt re s ults in the pumping o f pro to ns a cros s the inne r mitochondria l me mbra ne from

VII. Cha pte r S umma ry

81

Oxidative pho s pho rylatio n Oxidative pro c e s s e s , produce s uc h as the TCA c yc le and the β-o xidatio n o f fatty ac ids

NADH and FADH2

FMN, FAD-c o ntaining d e h yd ro g e n a s e s

dona te e le ctrons to

Co Q (c o e nzyme Q) Cyto c hro me bc 1

compris e d of

Ele c tro n trans po rt c hain

involve d in

Cyto c hro me c

le a ds to

Apo pto s is

Cyto c hro me a + a 3 (c yto c h ro m e o xid a s e )

Ele c tro n flo w Only c o mpo ne nt that c an re ac t dire c tly with o xyg e n

couple d with

Trans po rt o f pro to ns (H+) from

The matrix to the inte rme mbrane s pac e

vis ua lize d a s

cre a ting

An e le c tric al and a pH g radie nt

• Ric h in pro te in • Impe rme able to mo s t s mall mo le c ule s • Co ntains trans po rte rs fo r s pe c ific c o mpo unds

a cros s

The inne r mito c ho ndrial me mbrane

Nota ble be ca us e

a llowing

Pro to ns to re e nte r the mito c ho ndrial matrix by

Pas s ing thro ug h Fo c hanne l in the ATP s yn th a s e c o mple x (Co mple x V)

Re duc e d s ubs trate

Nota ble be ca us e

Ele c tro n trans po rt and pho s pho rylatio n are tig htly c o uple d pro c e s s e s , and the pro to n g radie nt is the c o mmo n inte rme diate . Inhibitio n o f o ne pro c e s s , thus , inhibits the o the r.

re s ulting in

Co nfo rmatio nal c hang e s in the F1 do main o f ATP s yn th a s e that allo w the s ynthe s is o f ATP fro m ADP + P i

NAD+

MITOCHONDRIAL MATRIX vis ua lize d a s

H2 O NADH 1 /2 O

2

Oxidize d s ubs trate Inne r mito c ho ndrial me mbrane

ADP + Pi

ATP

e–

Complex I

e–

Complex III

e–

Complex IV

Complex V

INTERMEMBRANE S PACE

Fig ure 6.17 Ke y conce pt ma p for oxida tive phos phoryla tion (OXP HOS ). [Note : Ele ctron (e – ) flow a nd ATP s ynthe s is a re e nvis ione d a s s e ts of inte rlocking ge a rs to e mpha s ize the ide a of coupling.] TCA = trica rboxylic a cid; NAD(H) = nicotina mide a de nine dinucle otide ; FAD(H2 ) = fla vin a de nine dinucle otide ; FMN = fla vin mononucle otide .

82

6. Bioe ne rge tics a nd Oxida tive P hos phoryla tion

the ma trix to the inte rme mbra ne s pa ce . This proce s s cre a te s e le c tric al a nd pH g radie nts a cros s the inne r mitochondria l me mbra ne . Afte r protons ha ve be e n tra ns fe rre d to the cytos olic s ide of the me mbra ne , the y re e nte r the ma trix by pa s s ing through the F o proton cha nne l in ATP s yn th a s e (Co mple x V), dis s ipa ting the pH a nd e le ctrica l gra die nts a nd ca us ing conforma tiona l cha nge s in the β s ubunits of F 1 tha t re s ult in the s ynthe s is of ATP from a de nos ine diphos pha te + inorga nic phos pha te . Ele c tro n trans po rt a nd pho s pho rylatio n a re tig htly c o uple d in oxida tive phos phoryla tion (OXP HOS , Figure 6.17). Inhibition of one proce s s inhibits the othe r. The s e proce s s e s ca n be unc o uple d by unc o upling pro te in-1 of the inne r mitochondria l me mbra ne of ce lls in brown fa t a nd by s ynthe tic compounds s uch a s 2,4-dinitro phe no l a nd as pirin , a ll of which dis s ipa te the proton gra die nt. In uncouple d mitochondria , the e ne rgy produce d by the tra ns port of e le ctrons is re le a s e d a s he at ra the r tha n be ing us e d to s ynthe s ize ATP . Muta tions in mito c ho ndrial DNA, which is ma te rna lly inhe rite d, a re re s pons ible for s ome ca s e s of mito c ho ndrial dis e as e s s uch a s Le be r he re ditary o ptic ne uro pathy . The re le a s e of cytochrome c into the cytopla s m a nd s ubs e que nt a ctiva tion of prote olytic ca s pa s e s re s ults in a poptotic ce ll de a th.

S tudy Que s tio ns Choos e the ONE be s t a ns we r. 6.1 2,4-Dinitrophe nol, a n uncouple r of oxida tive phosphoryla tion, wa s us e d a s a we ight-loss a ge nt in the 1930s. Re ports of fa ta l ove rdose s le d to its dis continua tion in 1939. Which of the following would most like ly be true conce rning individua ls ta king 2,4-dinitrophe nol? A. Ade nos ine triphos pha te le ve ls in the mitochondria a re gre a te r tha n norma l. B. Body te mpe ra ture is e le va te d a s a re s ult of hype rme ta bolis m. C. Cya nide ha s no e ffe ct on e le ctron flow. D. The proton gra die nt a cros s the inne r mitochondria l me mbra ne is gre a te r tha n norma l. E. The ra te of e le ctron tra ns port is a bnorma lly low. 6.2 Which of the following ha s the s tronge s t te nde ncy to ga in e le ctrons ? A. B. C. D. E.

Coe nzyme Q Cytochrome c Fla vin a de nine dinucle otide Nicotina mide a de nine dinucle otide Oxyge n

6.3 Expla in why a nd how the ma la te -a s pa rta te s huttle move s nicotina mide a de nine dinucle otide re ducing e q uiva le nts from the cytos ol to the mitochondria l ma trix. 6.4 Ca rbon monoxide binds to a nd inhibits Comple x IV of the e le ctron tra ns port cha in. Wha t e ffe ct, if a ny, s hould this re s pira tory inhibitor ha ve on phos phoryla tion of a de nosine diphospha te to a de nosine triphospha te ?

Corre ct a ns we r = B. Whe n phos phoryla tion is uncouple d from e le ctron flow, a de cre a s e in the proton gra die nt a cros s the inne r mitochondria l me mbra ne a nd, the re fore , impa ire d ATP s ynthe s is is e xpe cte d. In a n a tte mpt to compe ns a te for this de fe ct in e ne rgy ca pture , me ta bolis m a nd e le ctron flow to oxyge n is incre a s e d. This hype rme ta bolis m will be a ccompa nie d by e le va te d body te mpe ra ture be ca us e the e ne rgy in fue ls is la rge ly wa s te d, a ppe a ring a s he a t. The e le ctron tra ns port cha in will s till be inhibite d by cya nide .

Corre ct a ns we r = E. Oxyge n is the te rmina l a cce ptor of e le ctrons in the e le ctron tra ns port cha in (ETC). Ele ctrons flow down the ETC to oxyge n be ca us e it ha s the highe s t (mos t pos itive ) re duction pote ntia l (E 0 ). The othe r choice s pre ce de oxyge n in the ETC a nd ha ve lowe r E 0 va lue s .

The re is no tra ns porte r for nicotina mide a de nine dinucle otide (NADH) in the inne r mitochondria l me mbra ne . Howe ve r, NADH ca n be oxidize d to NAD+ by the cytopla s mic is ozyme of ma la te de hydroge na s e a s oxa loa ce ta te is re duce d to ma la te . The ma la te is tra ns porte d a c ro s s th e in n e r m e m b ra n e , a n d th e m ito chondria l is ozyme of ma la te de hydroge na s e oxidiz e s it to oxa loa c e ta te a s mitochondria l NAD+ is re duce d to NADH. This NADH ca n be oxidize d by Comple x I of the e le ctron tra ns port cha in, ge ne ra ting thre e ATP through the couple d proce s s e s of oxida tive phos phoryla tion.

In h ib itio n o f th e e le ctro n tra n s po rt c ha in b y re s pira tory inhibitors s uch a s ca rbon monoxide re s ults in a n ina bility to ma inta in the proton gra die nt. P hos phoryla tion of ADP to ATP is , the re fore , inhibite d, a s a re a ncilla ry re a ctions s uch a s ca lcium upta ke by mitochondria , be ca us e the y a ls o re quire the proton gra die nt.

Intro duc tio n to Carbo hydrate s

7

I. OVERVIEW Ca rbohydra te s (s a ccha ride s ) a re the mos t a bunda nt orga nic mole cule s in na ture . The y ha ve a wide ra nge of functions , including providing a s ignifica nt fra ction of the die ta ry ca lorie s for mos t orga nis ms , a cting a s a s tora ge form of e ne rgy in the body, a nd s e rving a s ce ll me mbra ne compone nts tha t me dia te s ome forms of inte rce llula r communica tion. Ca rbohydra te s a ls o s e rve a s a s tructura l compone nt of ma ny orga nis ms , including the ce ll wa lls of ba cte ria , the e xos ke le ton of ma ny ins e cts , a nd the fibrous ce llulos e of pla nts . The e mpiric formula for ma ny of the s imple r ca rbohydra te s is (CH2 O)n , whe re n ≥ 3, he nce the na me “hydra te of ca rbon.”

II. CLAS S IFICATION AND S TRUCTURE Monos a ccha ride s (s imple s uga rs ) ca n be cla s s ifie d a ccording to the numbe r of ca rbon a toms the y conta in. Exa mple s of s ome monos a ccha ride s commonly found in huma ns a re lis te d in Figure 7.1. The y ca n a ls o be cla s s ifie d by the type of ca rbonyl group the y conta in. Ca rbohydra te s with a n a lde hyde a s the ir ca rbonyl group a re ca lle d a ldos e s , whe re a s thos e with a ke to a s the ir ca rbonyl group a re ca lle d ke tos e s (Figure 7.2). For e xa mple , glyce ra lde hyde is a n a ldos e , whe re a s dihydroxya ce tone is a ke tos e . Ca rbohydra te s tha t ha ve a fre e ca rbonyl group ha ve the s uffix –os e . [Note : Ke tos e s ha ve a n a dditiona l “ul” in the ir s uffix s uch a s xyulos e . The re a re e xce ptions , s uch a s fructos e , to this rule .] Monos a ccha ride s ca n be linke d by glycos idic bonds to cre a te la rge r s tructure s (Figure 7.3). Dis a ccha ride s conta in two monos a ccha ride units , oligos a ccha ride s conta in thre e to te n monos a ccha ride units, a nd polys a ccha ride s conta in more tha n te n monos a ccha ride units a nd ca n be hundre ds of s uga r units in le ngth.

3 4 5 6 7 9

Ge ne ric name s Ca rbons : trios e s Ca rbons : te tros e s Ca rbons : pe ntos e s Ca rbons : he xos e s Ca rbons : he ptos e s Ca rbons : nonos e s

Example s Glyce ra lde hyde Erythros e Ribos e Glucos e S e dohe ptulos e Ne ura minic a cid

Fig ure 7.1 Exa mple s of monos a ccha ride s found in huma ns , cla s s ifie d a ccording to the numbe r of ca rbons the y conta in.

A Alde hyde g ro up

B Ke to g ro up

A. Is o me rs and e pime rs Compounds tha t ha ve the s a me che mica l formula but ha ve diffe re nt s tructure s a re ca lle d is ome rs . For e xa mple , fructos e , glucos e , ma nnos e , a nd ga la ctos e a re a ll is ome rs of e a ch othe r, ha ving the s a me che mica l formula , C 6 H12 O 6 . Ca rbohydra te is ome rs tha t diffe r in configura tion a round only one s pe cific ca rbon a tom (with the e xce ption of the ca rbonyl ca rbon; s e e “a nome rs ” be low) a re de fine d a s e pime rs of e a ch othe r. For e xa mple , glucos e a nd ga la ctos e a re

CH2 OH Glyc e ralde hyde

Dihydro xyac e to ne

Fig ure 7.2 Exa mple s of a n a ldos e (A) a nd a ke tos e (B) s uga r.

83

84

7. Introduction to Ca rbohydra te s

Carbo n 4 o f g luc o s e

Carbo n 1 o f g alac to s e

HO

CH2 OH O

CH2 OH O O

OH

HOH

O OH

C-4 e pime rs be ca us e the ir s tructure s diffe r only in the pos ition of the –OH group a t ca rbon 4. [Note : The ca rbons in s uga rs a re numbe re d be ginning a t the e nd tha t conta ins the ca rbonyl ca rbon (tha t is , the a lde hyde or ke to group) a s s hown in Figure 7.4.] Glucos e a nd ma nnos e a re C-2 e pime rs . Howe ve r, be ca us e ga la ctos e a nd ma nnos e diffe r in the pos ition of –OH groups a t two ca rbons (ca rbons 2 a nd 4), the y a re is ome rs ra the r tha n e pime rs (s e e Figure 7.4). B. Enantio me rs

OH

Glyc o s idic bo nd

OH

Lac to s e : g alac to s yl-β(1→4)-g luc o s e

Fig ure 7.3 A glycos idic bond be twe e n two he xos e s producing a dis a ccha ride .

CHO

Galac to s e

C. Cyc lizatio n o f mo no s ac c haride s

HCOH HOCH H O CH HCOH CH2 OH C-4 e pime rs 1

CHO H 2C O H 3

Gluc o s e

HO C H H 4C O H H 5 C OH 6

CH2 OH

C-2 e pime rs CHO H O CH Manno s e

Is o me rs

HOCH HCOH HCOH CH2 OH

CH2 OH C O Fruc to s e

A s pe cia l type of is ome ris m is found in the pa irs of s tructure s tha t a re mirror ima ge s of e a ch othe r. The s e mirror ima ge s a re ca lle d e na ntiome rs , a nd the two me mbe rs of the pa ir a re de s igna te d a s a D- a nd a n L-s uga r (Figure 7.5). The va s t ma jority of the s uga rs in huma ns a re D-s uga rs . In the D is ome ric form, the –OH group on the a s ymme tric ca rbon (a ca rbon linke d to four diffe re nt a toms or groups ) fa rthe s t from the ca rbonyl ca rbon is on the right, whe re a s in the L-is ome r, it is on the le ft. Mos t e nzyme s a re s pe cific for e ithe r the D or the L form, but e nzyme s known a s ra ce ma s e s a re a ble to inte rconve rt D- a nd L-is ome rs .

HO C H H C OH H C OH CH2 OH

Fig ure 7.4 C-2 a nd C-4 e pime rs a nd a n is ome r of glucos e .

Le s s tha n 1% of e a ch of the monos a ccha ride s with five or more ca rbons e xis ts in the ope n-cha in (a cyclic) form in s olution. Ra the r, the y a re pre domina ntly found in a ring (cyclic) form, in which the a lde hyde (or ke to) group ha s re a cte d with a n a lcohol group on the s a me s uga r, ma king the ca rbonyl ca rbon (ca rbon 1 for a n a ldos e , ca rbon 2 for a ke tos e ) a s ymme tric. This a s ymme tric ca rbon is re fe rre d to a s the a nome ric ca rbon. 1. Ano me rs : Cre a tion of a n a nome ric ca rbon (the forme r ca rbonyl ca rbon), ge ne ra te s a ne w pa ir of is ome rs , the α a nd β configura tions of the s uga r (for e xa mple , α -D-glucopyra nos e a nd β-Dglucopyra nos e ; s e e Figure 7.6), tha t a re a nome rs of e a ch othe r. [Note : In the α configura tion, the –OH group on the a nome ric ca rbon proje cts to the s a me s ide a s the ring in a modifie d Fis che r proje ction formula (Figure 7.6A) a nd is tra ns to the CH2 OH group in a Ha worth proje ction formula (Figure 7.6B). The α a nd β forms a re not mirror ima ge s , a nd the y a re re fe rre d to a s dia s te re ome rs .] Enzyme s a re a ble to dis tinguis h be twe e n the s e two s tructure s a nd us e one or the othe r pre fe re ntia lly. For e xa mple , glycoge n is s ynthe s ize d from α -D-glucopyra nos e , whe re a s ce llulos e is s ynthe s ize d from β-D-glucopyra nos e . The cyclic α a nd β a nome rs of a s uga r in s olution s ponta ne ous ly (but s lowly) form a n e quilibrium mixture , a proce s s known a s muta rota tion (s e e Figure 7.6). [Note : For glucos e , the α form ma ke s up 36% of the mixture .] 2. Re duc ing s ug ars : If the hydroxyl group on the a nome ric ca rbon of a cyclize d s uga r is not linke d to a nothe r compound by a glycos idic bond, the ring ca n ope n. The s uga r ca n a ct a s a re ducing a ge nt a nd is te rme d a re ducing s uga r. S uch s uga rs ca n re a ct with chromoge nic a ge nts (for e xa mple , the Be ne dict re a ge nt) ca us ing the re a ge nt to be re duce d a nd colore d, with the a lde hyde group of the a cyclic s uga r be coming oxidize d. All monos a ccha ride s , but not a ll dis a ccha ride s , a re re ducing s uga rs . [Note :

II. Cla s s ifica tion a nd S tructure

85

Glucos e ca n ha ve its te rmina l hydroxyl group oxidize d to a ca rboxyl group, forming glucuronic a cid (s e e p. 161), or its a lde hyde group oxidize d to a hydroxyl group, forming a s uga r a lcohol.]

O CH H C HO OH C H H C HO H C HO OH C H2 e cos u l -G

A colorime tric te s t ca n de te ct a re ducing s uga r in urine . A pos itive re s ult is indica tive of a n unde rlying pa thology, be ca us e s uga rs a re not norma lly pre s e nt in urine , a nd ca n be followe d up by more s pe cific te s ts to ide ntify the re ducing s uga r.

CH O H C OH HO C H H C OH H C OH CH 2OH D-G lu c os e

L

D. Jo ining o f mo no s ac c haride s Monos a ccha ride s ca n be joine d to form dis a ccha ride s , oligos a ccha ride s , a nd polys a ccha ride s . Importa nt dis a ccha ride s include la ctos e (ga la ctos e + glucos e ), s ucros e (glucos e + fructos e ), a nd ma ltos e (glucos e + glucos e ). Importa nt polys a ccha ride s include bra nche d glycoge n (from a nima l s ource s ) a nd s ta rch (pla nt s ource s ) a nd unbra nche d ce llulos e (pla nt s ource s ). Ea ch is a polyme r of glucos e . The bonds tha t link s uga rs a re ca lle d glycos idic bonds . The s e a re forme d by e nzyme s known a s glycos yltra ns fe ra s e s tha t us e nucle otide s uga rs s uch a s uridine diphos pha te glucos e a s s ubs tra te s .

Fig ure 7.5 Ena ntiome rs (mirror ima ge s ) of glucos e . De s igna tion of D a nd L is by compa ris on to the trios e , glyce ra lde hyde . [Note : The a s ymme tric ca rbons a re s hown in gre e n.]

1. Naming g lyc o s idic bo nds : Glycos idic bonds be twe e n s uga rs a re na me d a ccording to the numbe rs of the conne cte d ca rbons a nd with re ga rd to the pos ition of the a nome ric hydroxyl group of the s uga r involve d in the bond. If this a nome ric hydroxyl is in the α configura tion, the linka ge is a n α -bond. If it is in the β configura tion, the linka ge is a β-bond. La ctos e , for e xa mple , is s ynthe s ize d by forming a glycos idic bond be twe e n ca rbon 1 of β-ga la ctos e a nd ca rbon 4 of glucos e . The linka ge is , the re fore , a β(1→4) glycos idic bond (s e e Figure 7.3). [Note : Be ca us e the a nome ric e nd of the glucos e re s idue is not involve d in the glycos idic linka ge , it (a nd, the re fore , la ctos e ) re ma ins a re ducing s uga r.] E. Co mple x c arbo hydrate s Ca rbohydra te s ca n be a tta che d by glycos idic bonds to nonca rbohydra te s tructure s , including purine a nd pyrimidine ba s e s (found in

A

HO

1C

H

H C OH HO C H O H C OH

O 1C

H

H 2C OH HO 3C H H 4C OH

H

1C

OH

H C OH HO C H O H C OH

H 5C

H 5C OH

H 5C

H C OH

H 6C OH

H C OH

H

H

β-D-Gluc o pyrano s e

D-Gluc o s e

B

Carbo nyl c arbo n

Ano me ric c arbo n

Ano me ric c arbo n HOCH2 H HO

5

O

OH

H OH

H

H

OH

1

H

HOCH2 OH H 5 H HC=O 1 OH H HO H H

OH

HOCH2 H HO

5

O

H

H OH

H

H

OH

1

OH

H

α-D-Gluc o pyrano s e

β-D-Gluc o pyrano s e

D-Gluc o s e

α-D-Gluc o pyrano s e

Fig ure 7.6 A The inte rconve rs ion (muta rota tion) of the α a nd β a nome ric forms of glucos e s hown a s modifie d Fis che r proje ction formula s . B. The inte rconve rs ion s hown a s Ha worth proje ction formula s . [Note : A s uga r with a s ix-me mbe re d ring (5C + 1O) is te rme d a pyra nos e , whe re a s one with a five -me mbe re d ring (4C + 1O) is a fura nos e . Virtua lly a ll glucos e in s olution is in the pyra nos e form.]

86

7. Introduction to Ca rbohydra te s

O H

O C CH2

+

OH

nucle ic a cids ), a roma tic rings (s uch a s thos e found in s te roids a nd bilirubin), prote ins (found in glycoprote ins a nd prote oglyca ns ), a nd lipids (found in glycolipids ), to form glycos ide s .

NH2

S ug ar

As parag ine

H2 O

Po lype ptide c hain

O H

O C CH2

H N

1. N- and O-g lyc o s ide s : If the group on the nonca rbohydra te mole cule to which the s uga r is a tta che d is a n –NH2 group, the s tructure is a n N-glycos ide , a nd the bond is ca lle d a n N-glycos idic link. If the group is a n –OH, the s tructure is a n O-glycos ide , a nd the bond is a n O-glycos idic link (Figure 7.7). [Note : All s uga r–s uga r glycos idic bonds a re O-type linka ge s .]

N-Glyc o s idic bo nd O H

III. DIGES TION OF DIETARY CARBOHYDRATES

OH

+ HO CH2

S e rine

S ug ar

Po lype ptide c hain

H2 O O H O

CH2

O-Glyc o s idic bo nd

Fig ure 7.7 Glycos ide s : e xa mple s of N- a nd O-glycos idic bonds .

The principa l s ite s of die ta ry ca rbohydra te dige s tion a re the mouth a nd inte s tina l lume n. This dige s tion is ra pid a nd is ca ta lyze d by e nzyme s known a s glycos ide hydrola s e s (glycos ida s e s ) tha t hydrolyze glycos idic bonds (Figure 7.8). Be ca us e the re is little monos a ccha ride pre s e nt in die ts of mixe d a nima l a nd pla nt origin, the e nzyme s a re prima rily e ndoglycos ida s e s tha t hydrolyze polys a ccha ride s a nd olios a ccha ride s , a nd dis a ccha rida s e s tha t hydrolys e tri- a nd dis a ccha ride s into the ir re ducing s uga r compone nts . Glycos ida s e s a re us ua lly s pe cific for the s tructure a nd configura tion of the glycos yl re s idue to be re move d a s we ll a s for the type of bond to be broke n. The fina l products of ca rbohydra te dige s tion a re the monos a ccha ride s , glucos e , ga la ctos e , a nd fructos e tha t a re a bs orbe d by ce lls of the s ma ll inte s tine . A. S alivary a -amylas e

O

H

O

H O

H2 O

Glycos ida s e O

H OH

H

O

HO

Fig ure 7.8 Hydrolys is of a glycos idic bond.

The ma jor die ta ry polys a ccha ride s a re of pla nt (s ta rch, compos e d of a mylos e a nd a mylope ctin) a nd a nima l (glycoge n) origin. During ma s tica tion, s a liva ry α -a myla s e a cts brie fly on die ta ry s ta rch a nd glycoge n, hydrolyzing ra ndom α(1→4) bonds . [Note : The re a re both α(1→4)- a nd β(1→4)-e ndoglucos ida s e s in na ture , but huma ns do not produce the la tte r. The re fore , we a re una ble to dige s t ce llulos e, a ca rbohydra te of pla nt origin conta ining β(1→4) glycos idic bonds be twe e n glucos e re s idue s .] Be ca us e bra nche d a mylope ctin a nd glycoge n a lso conta in α (1→6) bonds , which α-a myla s e ca nnot hydrolyze , the dige s t re s ulting from its a ction conta ins a mixture of s hort, bra nche d a nd unbra nche d oligos a ccha ride s known a s de xtrins (Figure 7.9). [Note : Dis a ccha ride s a re a ls o pre s e nt a s the y, too, a re res is ta nt to a myla s e.] Ca rbohydra te dige s tion ha lts te mpora rily in the s toma ch, be ca us e the high a cidity ina ctiva te s s a liva ry α-a myla s e. B. Panc re atic a -amylas e Whe n the a cidic s toma ch conte nts re a ch the s ma ll inte s tine , the y a re ne utra lize d by bica rbona te s e cre te d by the pa ncre a s , a nd pa ncre a tic α -a myla s e continue s the proce s s of s ta rch dige s tion. C. Inte s tinal dis ac c haridas e s The fina l dige s tive proce s s e s occur prima rily a t the mucos a l lining of the uppe r je junum a nd include the a ction of s e ve ra l dis a ccha rida s e s (s e e Figure 7.9). For e xa mple , is oma lta s e cle a ve s the α (1→6)

III. Dige s tion of Die ta ry Ca rbohydra te s bond in is oma ltos e , a nd ma lta s e cle a ve s the α (1→4) bond in ma ltos e a nd ma ltotrios e , e a ch producing glucos e . S ucra s e cle a ve s the α(1→2) bond in s ucros e , producing glucos e a nd fructos e , a nd la cta s e (β-ga la ctos ida s e ) cle a ve s the β(1→4) bond in la ctos e , producing ga la ctos e a nd glucos e . [Note : The s ubs tra te s for is oma lta s e a re broa de r tha n its na me s ugge s ts , a nd it hydrolyze s the ma jority of ma ltos e .] Tre ha los e , a n α (1→1) dis a ccha ride of glucos e found in mus hrooms a nd othe r fungi is cle a ve d by tre ha la s e . The s e e nzyme s a re tra ns me mbra ne prote ins of the brus h borde r on the lumina l s urfa ce of the inte s tina l mucos a l ce lls .

87

MOUTH

S a liva ry α -a myla s e

S tarc h Lac to s e S uc ro s e Ce llulo s e

D. Inte s tinal abs o rptio n o f mo no s ac c haride s The duode num a nd uppe r je junum a bs orb the bulk of the monos a ccha ride products of dige s tion. Howe ve r, diffe re nt s uga rs ha ve diffe re nt me cha nis ms of a bs orption (Figure 7.10). For e xa mple , ga la ctos e a nd glucos e a re tra ns porte d into the mucos a l ce lls by a n a ctive , e ne rgy-de pe nde nt proce s s tha t re quire s a concurre nt upta ke of s odium ions , a nd the tra ns port prote in is the s odium-de pe nde nt glucos e cotra ns porte r 1 (S GLT-1). Fructos e utilize s a n e ne rgy- a nd s odium-inde pe nde nt monos a ccha ride tra ns porte r (GLUT-5) for its a bs orption. All thre e monos a ccha ride s a re tra ns porte d from the inte s tina l mucos a l ce ll into the porta l circula tion by ye t a nothe r tra ns porte r, GLUT-2. (S e e p. 97 for a dis cus s ion of the s e tra ns porte rs .)

Lo w pH s to ps ac tio n o f s a liva ry α -a m yla s e

S TOMACH

S MALL INTES TINE

S ucra s e a nd is oma lta s e a re e nzymic a ctivitie s of a s ingle prote in (SI ) which is cle a ve d into two functiona l s ubunits tha t re ma in a s s ocia te d in the ce ll me mbra ne , forming the s ucra s e -is oma lta s e comple x. In contra s t, ma lta s e is one of two e nzymic a ctivitie s of a s ingle me mbra ne prote in ma lta s e -glucoa myla s e (MGA) tha t doe s not ge t cle a ve d. Its s e cond e nzymic a ctivity, glucoa myla s e , cle a ve s α (1→4) glycos idic bonds in de xtrins .

S tarc h de xtrins Is o malto s e Malto s e Malto trio s e Lac to s e S uc ro s e Ce llulo s e

PANCREAS

to LIVER

Is o malto s e Malto s e Malto trio s e Lac to s e S uc ro s e

Po rtal c irc ulatio n

Muc o s al c e ll me mbrane – bo und e nzyme s Gluc o s e Is oma lta s e Fruc to s e Ma lta s e Galac to s e La cta s e S ucras e Tre ha la s e

Ce llulo s e

Fig ure 7.9 Dige s tion of ca rbohydra te s . [Note : Indige s tible ce llulos e e nte rs the colon a nd is e xcre te d.] Gluc o s e Fruc to s e Galac to s e

Na + S GLT-1

GLUT-5

Brus h borde r on lumina l s urfa ce

E. Abno rmal de g radatio n o f dis ac c haride s The ove ra ll proce s s of ca rbohydra te dige s tion a nd a bs orption is s o e fficie nt in he a lthy individua ls tha t ordina rily a ll dige s tible die ta ry ca rbohydra te is a bs orbe d by the time the inge s te d ma te ria l re a che s the lowe r je junum. Howe ve r, be ca us e only monos a ccha ride s a re a bs orbe d, a ny de ficie ncy (ge ne tic or a cquire d) in a s pe cific dis a ccha rida s e a ctivity of the inte s tina l mucos a ca us e s the pa s s a ge of undige s te d ca rbohydra te into the la rge inte s tine . As a cons e que nce of the pre s e nce of this os motica lly a ctive ma te ria l, wa te r is dra wn from the mucos a into the la rge inte s tine , ca us ing os motic dia rrhe a . This is re inforce d by the ba cte ria l fe rme nta tion of the re ma ining ca rbohydra te to two- a nd thre e -ca rbon compounds (which a re a ls o os motica lly a ctive ) plus la rge volume s of CO 2 a nd H2 ga s , ca us ing a bdomina l cra mps , dia rrhe a , a nd fla tule nce .

P a ncre a tic α -a myla s e

Na + Fruc to s e Gluc o s e Galac to s e

Circula tion

ATP

2K+ ADP + Pi

3Na + Na +−K+ ATP a s e (Na +−K+ pump)

2K+

GLUT-2

Fig ure 7.10 Abs orption by inte s tina l mucos a l ce lls of the monos a ccha ride products of ca rbohydra te dige s tion. S GLT-1 = s odium-de pe nde nt glucos e tra ns porte r.

88

7. Introduction to Ca rbohydra te s

La c ta s e de fic ie nc y S MALL INTES TINE Lac to s e

Galac to s e + Gluc o s e

LARGE INTES TINE Lac to s e

H2 c an be me as ure d in the bre ath.

BACTERIA

Two -Carbo n me tabo lite s CO2 (s uc h as ac e tic ac id)

H2 Thre e -Carbo n me tabo lite s (s uc h as lac tic ac id)

BLOATING DIARRHEA DEHYDRATION

Fig ure 7.11 Abnorma l la ctos e me ta bolis m. H2 = hydroge n ga s .

H2 O

1. Dig e s tive e nzyme de fic ie nc ie s : Ge ne tic de ficie ncie s of the individua l dis a ccha rida s e s re s ult in dis a ccha ride intole ra nce . Alte ra tions in dis a ccha ride de gra da tion ca n a ls o be ca us e d by a va rie ty of inte s tina l dis e a s e s , ma lnutrition, a nd drugs tha t injure the mucos a of the s ma ll inte s tine . For e xa mple , brus h borde r e nzyme s a re ra pidly los t in norma l individua ls with s e ve re dia rrhe a , ca us ing a te mpora ry, a cquire d e nzyme de ficie ncy. The re fore , pa tie nts s uffe ring or re cove ring from s uch a dis orde r ca nnot drink or e a t s ignifica nt a mounts of da iry products or s ucros e without e xa ce rba ting the dia rrhe a . 2. Lac to s e into le ranc e : More tha n 70% of the world’s a dults a re la ctos e intole ra nt (Figure 7.11). This is pa rticula rly ma nife s te d in ce rta in popula tions . For e xa mple , up to 90% of a dults of Africa n or As ia n de s ce nt a re la cta s e -de ficie nt a nd, the re fore , a re le s s a ble to me ta bolize la ctos e tha n individua ls of Northe rn Europe a n origin. The a ge -de pe nde nt los s of la cta s e a ctivity re pre s e nts a re duction in the a mount of e nzyme produce d. It is thought to be ca us e d by s ma ll va ria tions in the DNA s e que nce of a re gion on chromos ome 2 tha t controls e xpre s s ion of the ge ne for la cta s e , a ls o on chromos ome 2. Tre a tme nt for this dis orde r is to re duce cons umption of milk a nd e a t yogurts a nd s ome che e s e s (ba cte ria l a ction a nd a ging proce s s de cre a s e la ctos e conte nt) a s we ll a s gre e n ve ge ta ble s , s uch a s broccoli, to e ns ure a de qua te ca lcium inta ke ; to us e la cta s e -tre a te d products ; or to ta ke la cta s e in pill form prior to e a ting. [Note : Be ca us e the los s of la cta s e is the norm for mos t of the world’s a dults , us e of the te rm “a dult hypola cta s ia ” for la ctos e intole ra nce is be coming more common.] Ra re ca s e s of conge nita l la cta s e de ficie ncy a re known. 3. Co ng e nital s uc ras e -is o maltas e de fic ie nc y: This a utos oma l re ce s s ive dis orde r re s ults in a n intole ra nce of inge s te d s ucros e . Conge nita l s ucra s e -is oma lta s e de ficie ncy ha s a pre va le nce of 0.02% in individua ls of Europe a n de s ce nt a nd a ppe a rs to be much more common in the Inuit pe ople of Gre e nla nd a nd Ca na da . Tre a tme nt include s the die ta ry re s triction of s ucros e a nd e nzyme re pla ce me nt the ra py. 4. Diag no s is : Ide ntifica tion of a s pe cific e nzyme de ficie ncy ca n be obta ine d by pe rforming ora l tole ra nce te s ts with the individua l dis a ccha ride s . Me a s ure me nt of hydroge n ga s in the bre a th is a re lia ble te s t for de te rmining the a mount of inge s te d ca rbohydra te not a bs orbe d by the body, but which is me ta bolize d ins te a d by the inte s tina l flora (s e e Figure 7.11).

IV. CHAPTER S UMMARY Mo no s ac c haride s (Figure 7.12) conta ining a n a lde hyde group a re ca lle d aldo s e s , a nd thos e with a ke to group a re ca lle d ke to s e s . Dis ac c haride s , o lig o s ac c haride s , a nd po lys ac c haride s cons is t of monos a ccha ride s linke d by g lyc o s idic bo nds . Compounds with the s a me che mica l formula but diffe re nt s tructure s a re ca lle d is o me rs . If

IV. Cha pte r S umma ry

89

two monos a ccha ride is ome rs diffe r in configura tion a round one s pe cific ca rbon a tom (with the e xce ption of the ca rbonyl ca rbon), the y a re de fine d a s e pime rs of e a ch othe r. If a pa ir of s uga rs a re mirror ima ge s (e nantio me rs ), the two me mbe rs of the pa ir a re de s igna te d a s D- a nd L-s ug ars . If the a lde hyde group on a n a cyclic s uga r ge ts oxidize d a s a chromoge nic a ge nt ge ts re duce d, tha t s uga r is a re ducing s uga r. Whe n a s uga r cyclize s , a n ano me ric c arbo n is cre a te d from the a lde hyde group of a n a ldos e or ke to group of a ke tos e . The s uga r ca n ha ve two configura tions , a or β. A s uga r with its a nome ric ca rbon linke d to a nothe r s tructure forms a g lyc o s ide . S uga rs ca n be a tta che d e ithe r to a n –NH2 or a n –OH group, producing N- a nd O-g lyc o s ide s . S a liva ry α-a m yla s e a cts on die tary po lys ac c haride s (s ta rch, glycoge n), producing oligos a ccha ride s . P a n c re a tic α-a m yla s e continue s the proce s s of ca rbohydra te dige s tion.The fina l dige s tive proce s s e s occur a t the muc o s al lining of the s mall inte s tine . S e ve ra l dis a ccha rida s e s (for e xa mple , la c ta s e [β-g a la c to s id a s e ], s u c ra s e , is o m a lta s e , a nd m a lta s e ) produce monos a ccha ride s (glucos e , ga la ctos e , a nd fructos e ). The s e e nzyme s a re trans me mbrane pro te ins of the lumina l brus h bo rde r of inte s tinal muc o s al c e lls . Abs orption of the monos a ccha ride s re quire s s pe cific tra ns porte rs . If ca rbohydra te de gra da tion is de ficie nt (a s a re s ult of he re dity, dis e a s e , or drugs tha t injure the inte s tina l mucos a ), undige s te d ca rbohydra te will pa s s into the la rge inte s tine , whe re it ca n ca us e o s mo tic diarrhe a. Ba cte ria l fe rme nta tion of the ma te ria l produce s la rge volume s of CO 2 a nd H2 , ca us ing a bdomina l cra mps , dia rrhe a , a nd fla tule nce . Lac to s e into le ranc e , prima rily ca us e d by the a ge -de pe nde nt los s of la c ta s e (adult hypo lac tas ia), is by fa r the mos t common of the s e de ficie ncie s .

Mo no s ac c haride s (s imple s ug ars )

Die tary c arbo hydrate s

ca n be compa re d with othe r monos a ccha ride s a nd cla s s ifie d a s

ca n be cla s s ifie d a s

a re compris e d of ca n link to form

Aldo s e s

Ke to s e s

if the y conta in

if the y conta in

Alde hyde g ro up H–C=O

Ke to g ro up C=O

Is o me rs

S ame c he mic al fo rmula

s uga r is cla s s ifie d a s Re duc ing s ug ar

Enantio me rs if the y a re

Diffe r in c o nfig uratio n aro und o ne s pe c ific c arbo n ato m

if ha s

Ring o pe ns and alde hyde g ro up o f the ac yc lic s ug ar g e ts o xidize d

Epime rs if the y

Ano me ric c arbo n

the n

for e xa mple

if the y ha ve

ma y cyclize to produce a n

Hydro xyl g ro up no t attac he d to an o the r mo le c ule

Po ly-, o lig o -, and dis ac c haride s

ca n be

Mirro r imag e s o f e ac h o the r

Co vale ntly attac he d to ano the r mo e c ule is cla s s ifie d a s N-Glyc o s idic linkag e O-Glyc o s idic linkag e

if a tta che d to –NH2 g ro up –OH g ro up

if a tta che d to

Dis ac c haride s

dige s te d by hydrola s e s which produce

Mo no s ac c haride s • Glucos e • Ga la ctos e • Fructos e

tha t a re a bs orbe d by

For e xa mple : S ucros e = glucos e + fructos e La ctos e = ga la ctos e + glucos e Ma ltos e = glucos e + glucos e

Inte s tinal muc o s al c e lls

Olig o s ac c haride s Po lys ac c haride s ca n be

Line ar For e xa mple : a mylos e

Branc he d For e xa mple : glycoge n

Fig ure 7.12 Ke y conce pt ma p for the cla s s ifica tion a nd s tructure of monos a ccha ride s a nd the dige s tion of die ta ry ca rbohydra te s .

90

7. Introduction to Ca rbohydra te s

S tudy Que s tio n Choos e the ONE be s t a ns we r. 7.1 Which of the following s ta te me nts be s t de s cribe s glucos e ? A. It is a C-4 e pime r of ga la ctos e . B. It is a ke tos e a nd us ua lly e xis ts a s a fura nos e ring in s olution. C. It is produce d from die ta ry s ta rch by the a ction of α -a myla s e . D. It is u tilize d in b iolog ica l s ys te m s o nly in the L-is ome ric form. 7.2 A young ma n e nte re d his phys icia n’s office compla ining of bloa ting a nd dia rrhe a . His e ye s we re s unke n, a nd the phys icia n note d a dditiona l s igns of de hydra tion. The pa tie nt’s te mpe ra ture wa s norma l. He e xpla ine d tha t the e pis ode ha d occurre d following a birthda y pa rty a t which he ha d pa rticipa te d in a n ice cre a m–e a ting conte s t. The pa tie nt re porte d prior e pis ode s of a s imila r na ture following inge s tion of a s ignifica nt a mount of da iry products . This clinica l picture is mos t proba bly due to a de ficie ncy in the a ctivity of: A. B. C. D. E.

Corre ct a ns we r = A. Glucos e a nd ga la ctos e diffe r only in configura tion a round ca rbon 4 a nd s o a re C-4 e pime rs tha t a re inte rconve rtible by the a ction of a n e pime ra s e . Glucos e is a n a ldos e s uga r tha t typica lly e xis ts a s a pyra nos e ring in s olution. Fructos e , howe ve r, is a ke tos e with a fura nos e ring. α -Amyla s e doe s not produce monos a ccha ride s . The D-is ome ric form of ca rbohydra te s is mos t typica lly the form found in biologic s ys te ms , in contra s t to a mino a cids .

Corre ct a ns we r = B. The phys ica l s ymptoms s ugge s t a de ficie ncy in a n e nzyme re s pons ible for ca rbohydra te de gra da tion. The s ymptoms obs e rve d following the inges tion of da iry products sugges t that the patient is deficie nt in lactase .

is oma lta s e . la cta s e . pa ncre a tic α -a myla s e . s a liva ry α -a myla s e . s ucra s e .

7.3 Routine e xa mina tion of the urine of a n a s ymptoma tic pe dia tric pa tie nt s howe d a pos itive re a ction with Clinite s t (a coppe r re duction me thod of de te cting re ducing s uga rs ) but a ne ga tive re a ction with the glucos e oxida s e te s t for de te cting glucos e . Us ing the s e da ta , s how on the cha rt be low which of the s uga rs could (YES ) or could not (NO) be pre s e nt in the urine of this individua l. S UGAR

YES

NO

Fructos e

Ea ch of the lis te d s uga rs , e xce pt for s ucros e a nd glucos e , could be pre s e nt in the urine of this individua l. Clinite s t is a nons pe cific te s t tha t produce s a cha nge in color if urine is pos itive for re ducing s ubs ta nce s s uch a s re ducing s uga rs (fructos e , ga la ctos e , glucos e , la ctos e , xylulos e ). Be ca us e s ucros e is not a re ducing s uga r, it is not de te cte d by Clinite s t. The glucos e oxida s e te s t will de te ct only glucos e , a nd it ca nnot de te ct othe r s uga rs . The ne ga tive glucos e oxida s e te s t in the fa ce of a pos itive re ducing s uga r te s t me a ns tha t glucos e ca nnot be the re ducing s uga r in the pa tie nt’s urine .

Ga la ctos e Glucos e La ctos e S ucros e Xylulos e 7.4 Why a re α -glucos ida s e inhibitors tha t a re ta ke n with me a ls , s uch a s a ca rbos e a nd miglitol, us e d in the tre a tme nt of dia be te s ? Wha t e ffe ct s hould the s e drugs ha ve on the dige s tion of la ctos e ?

α -Glucos ida s e inhibitors s low the production of glucos e from die ta ry ca rbohydra te s , the re by re ducing the pos tpra ndia l ris e in blood glucos e a nd fa cilita ting be tte r blood glucos e control in dia be tics . The s e drugs ha ve no e ffe ct on la ctos e dige s tion be ca us e the dis a ccha ride la ctos e contains a β-glycosidic bond, not a n α -glycos idic bond.

tahir99-VRG & vip.persianss.ir

Intro duc tio n to Me tabo lis m and Glyc o lys is I. INTRODUCTION TO METABOLIS M In Chapter 5, individua l enzymic reactions we re analyzed in an effort to explain the me cha nisms of ca ta lys is. Howe ver, in cells, the s e re a ctions rarely occur in isolation but, rather, are organized into multistep sequences called pathways, such as that of glycolysis (Figure 8.1). In a pathway, the product of one reaction serves as the substrate of the subsequent reaction. Different pathways can also intersect, forming an integrated and purposeful network of chemical reactions. These are collectively called metabolism, which is the sum of all the chemical changes occurring in a cell, a tissue, or the body. Most pathways can be classified as either catabolic (degrada tive ) or ana bolic (s ynthe tic). Ca ta bolic re actions bre ak down comple x molecules, such as proteins, polysaccharides, and lipids, to a few simple molecules (for example, CO 2, NH3 [ammonia], and H2O). Anabolic pathways form complex end products from simple precursors, for example, the synthesis of the polysaccharide, glycogen, from glucose. [Note: Pathways that regenerate a component are called cycles.] In the following chapters, this te xt focuse s on the centra l metabolic pa thways tha t a re involve d in synthesizing and degrading carbohydrates, lipids, and amino acids.

8 The pro duc t o f o ne re ac tio n is the s ubs trate o f the s ubs e que nt re ac tio n. Glucos e 6-P

Glucos e Gluc

Fructos e 6-P Fructos e 1,6-bis phos pha te Glyce ra lde hyde 3-P

Dihydroxya ce tone -P

1,3-Bis phos phoglyce ra te 3-P hos phoglyce ra te 2-P hos phoglyce ra te P hos phoe nolpyruva te La cta te

P yruva te

A. Me tabo lic map It is conve nie nt to inve s tiga te me ta bolis m by e xa mining its compone nt pa thwa ys . Ea ch pa thwa y is compos e d of multie nzyme s e que nce s , a nd e a ch e nzyme , in turn, ma y e xhibit importa nt ca ta lytic or re gula tory fe a ture s . To provide the re a de r with the “big picture ,” a me ta bolic ma p conta ining the importa nt ce ntra l pa thwa ys of ene rgy me ta bolis m is pre s e nte d in Figure 8.2. This ma p is us e ful in tra cing conne ctions be twe e n pa thwa ys , vis ua lizing the purpos e ful “move me nt” of me ta bolic inte rme dia te s , a nd de picting the e ffe ct on the flow of inte rme dia te s if a pa thwa y is blocke d (for e xa mple , by a drug or a n inhe rite d de ficie ncy of a n e nzyme ). Throughout the ne xt thre e units of this book, e a ch pa thwa y unde r dis cus s ion will be re pe a te dly fea ture d a s pa rt of the ma jor me ta bolic ma p s hown in Figure 8.2.

Fig ure 8.1 Glycolys is , a n e xa mple of a me ta bolic pa thwa y. [Note : P yruva te to phos phoe nolpyruva te re quire s two re a ctions .] Curve d re a ction a rrows ( ) indica te forwa rd a nd re ve rs e re a ctions tha t a re ca ta lyze d by diffe re nt e nzyme s .P = phos pha te .

B. Catabo lic pathways Ca ta bolic re a ctions s e rve to ca pture che mica l e ne rgy in the form of a de nos ine triphos pha te (ATP ) from the de gra da tion of e ne rgy-rich fue l mole cule s . Ca ta bolis m a ls o a llows mole cule s in the die t (or nutrie nt mole cule s s tore d in ce lls ) to be conve rte d into building blocks ne e de d for the s ynthe s is of comple x mole cule s . Energy ge ne ra tion by 91 tahir99-VRG & vip.persianss.ir

92

8. Introduction to Me ta bolis m a nd Glycolys is

Ribulo s e 5-P

6-P g luc o no lac to ne

Pe nto s e pho s phate pathway

Ribo s e 5-P

Galac to s e

Glyc o g e n

6-P g luc o nate

UDP-Gluc o s e

Galac to s e 1-P

Gluc o s e 1-P

UDP-Galac to s e

Xylulo s e 5-P

Gluc o s e 6-P

Gluc o s e

S e do he ptulo s e 7-P Erythro s e 4-P

Fruc to s e 6-P

Fruc to s e

Fruc to s e 1,6-bis -P Glyc e ralde hyde 3-P

Glyc e ralde hyde

Fruc to s e 1-P

Dihydro xyac e to ne -P

Glyc e ralde hyde 3-P Glyc o lys is

Glyc e ro l-P

1,3-Bis pho s pho g lyc e rate

Glyc e ro l

3-Pho s pho g lyc e rate Triac ylg lyc e ro l 2-Pho s pho g lyc e rate Fatty ac yl Co A Alanine Cys te ine Glyc ine S e rine Thre o nine NH3

Pho s pho e no lpyruvate Lac tate CO2

CO2

Pyruvate CO2

Malo nyl Co A

Ac e tyl Co A As parag ine

Fatty ac ids Triac ylg lyc e ro l s ynthe s is and de g radatio n

Ac e to ac e tate Is o le uc ine

Carbamo yl-P

Le uc ine Phe nylalanine Tyro s ine Trypto phan Lys ine

β-Hydro xybutyrate As partate

Oxalo ac e tate

Citrate

Citrulline

Ornithine

Arg inino s uc c inate Ure a c yc le Arg inine

Malate Tric arbo xylic Is o c itrate ac id (TCA) CO2 c yc le α-Ke to g lutarate Fumarate

Glutamine Glutamate

CO2 S uc c inate

S uc c inyl Co A

Pro line His tidine Arg inine

Me thylmalo nyl Co A

Ure a Phe nylalanine Tyro s ine

Is o le uc ine Me thio nine Valine Thre o nine

Pro pio nyl Co A Ac e tyl Co A Fatty ac yl Co A (o dd numbe r o f c arbo ns )

Fig ure 8.2 Importa nt re a ctions of inte rme dia ry me ta bolis m. S e ve ra l importa nt pa thwa ys to be dis cus s e d in la te r cha pte rs a re highlighte d. Curve d re a ction a rrows ( ) indica te forwa rd a nd re ve rs e re a ctions tha t a re ca ta lyze d by diffe re nt e nzyme s . The s tra ight a rrows ( ) indica te forwa rd a nd re ve rs e re a ctions tha t a re ca ta lyze d by the s a me e nzyme . Blue te xt = inte rme dia te s of ca rbohydra te me ta bolis m; brown text = inte rme dia te s of lipid me ta bolis m; green text = inte rme dia te s of prote in me ta bolis m. UDP = uridine diphos pha te ; P = phos pha te ; CoA = coe nzyme A.

tahir99-VRG & vip.persianss.ir

II. Re gula tion of Me ta bolis m

S tag e I:

93

Pro te ins

Carbo hydrate s

Fats

Hydro lys is o f c o mple x mo le c ule s to the ir c o mpo ne nt building blo c ks

S tag e II:

Amino ac ids

Mo no s ac c haride s

Glyc e ro l, fatty ac ids

Co nve rs io n o f building blo c ks to ac e tyl Co A (o r o the r s imple inte rme diate s ) Ac e tyl Co A

S tag e III: Oxidatio n o f ac e tyl Co A; o xidative pho s pho rylatio n

TCA c yc le

ATP CO2

Fig ure 8.3 Thre e s ta ge s of ca ta bolis m. CoA = coe nzyme A; TCA = trica rboxylic a cid. de gra da tion of comple x mole cule s occurs in thre e s ta ge s a s s hown in Figure 8.3. [Note : Ca ta bolic pa thwa ys a re typically oxida tive , a nd re quire oxidize d coe nzyme s s uch a s nicotina mide a de nine dinucle otide (NAD+).] 1. Hydro lys is o f c o mple x mo le c ule s : In the firs t s ta ge , comple x mole cule s a re broke n down into the ir compone nt building blocks . For e xa mple , prote ins a re de gra de d to a mino a cids , polys a ccha ride s to monos a ccha ride s , a nd fa ts (tria cylglyce rols ) to fre e fa tty a cids a nd glyce rol. 2. Conve rs io n of building bloc ks to s imple inte rme diate s : In the s e cond s ta ge , the s e dive rs e building blocks a re furthe r de gra de d to a ce tyl coe nzyme A (CoA) a nd a fe w othe r s imple molecule s . S ome e ne rgy is ca pture d a s ATP, but the a mount is s ma ll compa re d with the e ne rgy produce d during the third s ta ge of ca ta bolis m. 3. Oxidatio n o f ac e tyl c o e nzyme A: The trica rboxylic a cid (TCA)

cycle (s e e p. 109) is the fina l common pa thwa y in the oxida tion of fue l mole cule s tha t produce a ce tyl CoA. Oxida tion of a ce tyl CoA ge ne ra te s la rge a mounts of ATP via oxida tive phos phoryla tion a s e le ctrons flow from NADH a nd fla vin a de nine dinucle otide (FADH2 ) to oxyge n (s e e p. 73).

Ene rg y-yie lding nutrie nts

Co mple x mo le c ule s

Carbo hydrate s Fats Pro te ins

Pro te ins Po lys ac c haride s Lipids Nuc le ic ac ids

C

A

A T A B O L

C. Anabo lic pathways Ana bolic re a ctions combine s ma ll mole cule s , s uch a s a mino a cids , to form comple x mole cule s s uch a s prote ins (Figure 8.4). Ana bolic re a ctions re quire e ne rgy (a re e nde rgonic), which is ge ne ra lly provide d by the hydrolys is of ATP to a de nos ine diphos pha te (ADP ) a nd inorga nic phos pha te (P i). Ana bolic re a ctions ofte n involve che mica l re ductions in which the re ducing powe r is mos t fre que ntly provide d by the e le ctron donor NADP H (s e e p. 147). Note tha t ca ta bolis m is a conve rge nt proce s s (tha t is , a wide va rie ty of mole cule s a re tra ns forme d into a fe w common e nd products ). By contra s t, a na bolis m is a dive rge nt proce s s in which a fe w bios ynthe tic pre curs ors form a wide va rie ty of polyme ric, or comple x, products .

I S M

Che mic al e ne rg y ATP NADH

N A B O L I S M

Ene rg y-po o r e nd pro duc ts

Pre c urs o r mo le c ule s

CO2 H2 O NH3

Amino ac ids S ug ars Fatty ac ids Nitro g e no us bas e s

Fig ure 8.4 Compa ris on of ca ta bolic a nd a na bolic pa thwa ys . ATP = a de nos ine triphos pha te ; NADH = nicotina mide a de nine dinucle otide .

II. REGULATION OF METABOLIS M The pa thwa ys of me ta bolis m mus t be coordina te d s o tha t the production of e ne rgy or the s ynthe s is of e nd products me e ts the ne e ds of the

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8. Introduction to Me ta bolis m a nd Glycolys is

S ynaptic s ig naling Targ e t c e ll

Ne rve c e ll

Ne uro trans mitte r

A. Intrac e llular c o mmunic atio n

Endo c rine s ig naling Hormo ne Targ e t c e ll Blo o d ve s s e l

Dire c t c o ntac t Gap junc tio n

S ig naling c e ll

ce ll. Furthe rmore , individua l ce lls do not function in is ola tion but, ra the r, a re pa rt of a community of inte ra cting tis s ue s . Thus , a s ophis tica te d communica tion s ys te m ha s e volve d to coordina te the functions of the body. Re gula tory s igna ls tha t inform a n individua l ce ll of the me ta bolic s ta te of the body a s a whole include hormone s , ne urotra ns mitte rs , a nd the a va ila bility of nutrie nts . The s e , in turn, influe nce s igna ls ge ne ra te d within the ce ll (Figure 8.5).

Targ e t c e lls

Fig ure 8.5 S ome commonly us e d me cha nis ms for tra ns mis s ion of re gula tory s igna ls be twe e n ce lls .

The ra te of a me ta bolic pa thwa y ca n re s pond to re gula tory s igna ls tha t a ris e from within the ce ll. For e xa mple , the ra te of a pa thwa y ma y be influe nce d by the a va ila bility of s ubs tra te s , product inhibition, or a lte ra tions in the le ve ls of a llos te ric a ctiva tors or inhibitors . The s e intra ce llula r s igna ls typica lly e licit ra pid re s pons e s , a nd a re importa nt for the mome nt-to-mome nt re gula tion of me ta bolis m. B. Inte rc e llular c o mmunic atio n The ability to respond to intercellular signals is essential for the developme nt a nd s urviva l of orga nis ms . Signa ling be twe e n ce lls provide s for long-ra nge inte gra tion of me ta bolis m a nd us ua lly re s ults in a re s pons e, s uch a s a cha nge in ge ne expre ss ion, tha t is slowe r tha n is s e e n with intra ce llula r s igna ls . Communica tion be twe e n ce lls ca n be mediated, for example, by surface-to-surface contact and, in some tissues, by formation of ga p junctions , allowing dire ct communica tion between the cytoplasms of adjacent cells. However, for energy metabolism, the most importa nt route of communication is chemical signaling be twe e n ce lls by bloodborne hormone s or by ne urotra ns mitte rs . C. S e c o nd me s s e ng e r s ys te ms

The e xtrac e llular do main c o ntains the binding s ite fo r a lig and (a ho rmo ne o r ne uro trans mitte r).

Hormone s or ne urotra ns mitte rs ca n be thought of a s s igna ls a nd the ir re ce ptors a s s igna l de te ctors . Ea ch compone nt s e rve s a s a link in the communica tion be twe e n e xtra ce llula r e ve nts a nd che mica l cha nge s within the ce ll. Ma ny re ce ptors s igna l the ir re cognition of a bound liga nd by initia ting a s e rie s of re a ctions tha t ultima te ly re s ult in a s pe cific intra ce llula r re s pons e . “S e cond me s s e nge r” mole cule s , s o na me d be ca us e the y inte rve ne be twe e n the origina l me ss e nge r (the ne urotra ns mitte r or hormone ) a nd the ultima te e ffe ct on the ce ll, a re pa rt of the ca s ca de of e ve nts tha t tra ns la te s (tra ns duce s ) hormone or ne urotra ns mitte r binding into a ce llula r re s pons e . Two of the mos t wide ly re cognize d s e cond me s s e nge r s ys te ms a re the ca lcium/ phos pha tidylinos itol s ys te m (s e e p. 205) a nd the a de nylyl cycla s e (a de nyla te cycla s e ) s ys te m, which is pa rticula rly importa nt in re gula ting the pa thwa ys of inte rme dia ry me ta bolis m. D. Ade nylyl c yc las e

The intrac e llular do main inte rac ts with G pro te ins .

No te the s e ve n trans me mbrane α he llic e s .

Fig ure 8.6 S tructure of a typica l G prote in– couple d re ce ptor of the pla s ma me mbra ne .

The re cognition of a che mica l s igna l by s ome pla s ma (ce ll) me mbra ne re ce ptors , s uch a s the β- a nd α 2 -a dre ne rgic re ce ptors , trigge rs e ithe r a n incre a s e or a de cre a s e in the a ctivity of a de nylyl cycla s e (AC). This is a me mbra ne -bound e nzyme tha t conve rts ATP to 3',5'-a de nos ine monophos pha te (commonly ca lle d cyclic AMP , or cAMP ). The che mica l s igna ls a re mos t ofte n hormone s or ne urotra ns mitte rs , e a ch of which binds to a unique type of me mbra ne re ce ptor. The re fore , tis s ue s tha t re s pond to more tha n one che mica l s igna l mus t ha ve s e ve ra l diffe re nt re ce ptors , e a ch of which ca n be linke d to AC. The s e re ce ptors , known a s G prote in–couple d re ce ptors (GP CRs ), a re cha ra cte rize d by a n e xtra ce llula r liga nd-binding

tahir99-VRG & vip.persianss.ir

II. Re gula tion of Me ta bolis m doma in, s e ve n tra ns me mbra ne α he lice s , a nd a n intra ce llula r doma in tha t inte ra cts with G prote ins (Figure 8.6). 1. Guano s ine tripho s phate –de pe nde nt re g ulato ry pro te ins : The effect of the activated, occupied GPCR on second messenger formation is not direct but, rather, is mediated by spe cialized trime ric proteins (α, β, and γ subunits) of the ce ll membrane. These proteins, re fe rre d to a s G prote ins be ca us e the α s ubunit binds gua nine nucleotides (GTP and GDP), form a link in the chain of communication betwe en the receptor and AC. In the ina ctive form of a G protein, the α-subunit is bound to GDP (Figure 8.7). Binding of ligand ca uses a conformational change in the receptor, triggering repla cement of this GDP with GTP. The GTP-bound form of the α s ubunit dissociate s from the βγ subunits and moves to AC, which is thereby activate d. Ma ny mole cules of active Gα prote in are formed by one activated re ce ptor. [Note : The a bility of a hormone or ne urotra nsmitter to stimulate or inhibit AC de pends on the type of Gα protein that is linke d to the rece ptor. One type, designated G s , stimula tes AC, whe rea s a nother type, designated G i, inhibits the enzyme (not s hown in Figure 8.7).] The a ctions of the Gα –GTP comple x a re short-lived because Gα has an inherent GTPa se a ctivity, re sulting in the rapid hydrolysis of GTP to GDP. This causes inactiva tion of the Gα, its dissociation from AC, and re association with the βγ dimer.

95

1

Uno c c upie d re c e pto r do e s no t inte rac t with Gs pro te in. Ho rmo ne o r ne uro trans mitte r Ce ll me mbra ne Gs pro te in with bo und GDP

Extra ce llula r spa ce

Re c e pto r

β γ α Cytos ol

2

GDP

Inac tive a d e n ylyl c yc la s e

Oc c upie d re c e pto r c hang e s s hape and inte rac ts with α s ubunit o f Gs pro te in. Gs pro te in re le as e s GDP and binds GTP.

β γ α GTP

GTP

Toxins from Vibrio chole ra e (chole ra ) a nd Borde te lla pe rtus s is (whooping cough) ca us e ina ppropria te a ctiva tion of a de nylyl cycla s e through cova le nt modifica tion (ADP -ribos yla tion) of diffe re nt G prote ins . With chole ra , the GTPa s e a ctivity of Gα s is inhibite d in inte s tina l ce lls . With whooping cough, Gα i is ina ctiva te d in re s pira tory-tra ct ce lls .

3

GDP

α S ubunit o f Gs pro te in dis s o c iate s fro m βγ and ac tivate s a d e n ylyl c yc la s e . Ac tive a d e n ylyl c yc la s e ATP

β γ

α

2. Pro te in kinas e s : The ne xt ke y link in the cAMP s e cond me s s e n-

ge r s ys te m is the a ctiva tion by cAMP of a fa mily of e nzyme s ca lle d cAMP -de pe nde nt prote in kina s e s s uch a s prote in kina s e A (Figure 8.8). cAMP a ctiva te s prote in kina s e A by binding to its two re gula tory s ubunits , ca us ing the re le a s e of two a ctive , ca ta lytic s ubunits . The a ctive s ubunits ca ta lyze the tra ns fe r of phos pha te from ATP to s pe cific s e rine or thre onine re s idue s of prote in s ubs tra te s . The phos phoryla te d prote ins ma y a ct dire ctly on the ce ll’s ion cha nne ls or, if e nzyme s , ma y be come a ctiva te d or inhibite d. P rote in kina s e A ca n a ls o phos phoryla te prote ins tha t bind to DNA, ca us ing cha nge s in ge ne e xpre s s ion (s e e p. 456). [Note : S e ve ra l type s of prote in kina s e s a re not cAMP de pe nde nt, for e xa mple , prote in kina s e C de s cribe d on p. 205.] 3. Dephos phorylation of proteins : The phosphate groups added to proteins by protein kinases a re removed by protein phosphatases, e nzyme s tha t hydrolytica lly cle a ve phos pha te e s te rs (se e Figure 8.8). This ensures tha t changes in protein activity induced by phosphorylation a re not permanent. 4. Hydrolys is of cyc lic ade nos ine monophos phate : cAMP is ra pidly hydrolyze d to 5'-AMP by cAMP phos phodie s te ra s e , one of a family of enzymes that cleave the cyclic 3',5'-phosphodiester bond.

Inac tive a d e n ylyl c yc la s e

GTP

4

c AMP + PP i

Whe n ho rmo ne is no lo ng e r pre s e nt, the re c e pto r re ve rts to re s ting s tate . GTP o n the α s ubunit is hydro lyze d to GDP, α re jo ins βγ, and a d e n ylyl c yc la s e is de ac tivate d.

β γ α GDP

Inac tive a d e n ylyl c yc la s e

Pi

Fig ure 8.7 The re cognition of che mica l s igna ls by ce rta in me mbra ne re ce ptors trigge rs a n incre a s e (or, le s s ofte n, a de cre a s e ) in the a ctivity of a de nylyl cycla s e . GDP = gua nos ine diphos pha te ; GTP = gua nos ine triphos pha te ; cAMP = cyclic AMP .

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8. Introduction to Me ta bolis m a nd Glycolys is

Re g ulato ry s ubunits

R

C

R

C

5'-AMP is not a n intra ce llula r s igna ling mole cule . The re fore , the effects of neurotransmitter- or hormone-mediated increases of cAMP are rapidly terminated if the extracellular signal is removed. [Note: P hos phodie s te ra s e is inhibite d by the me thylxa nthine de riva tive , caffeine.]

Catalytic s ubunits

c AMP -d e p e n d e n t p ro te in kin a s e A ATP

Ade nylyl cycla s e

c AMP ( )

III. OVERVIEW OF GLYCOLYS IS The glycolytic pa thwa y is e mploye d by a ll tis s ue s for the oxida tion of glucos e to provide e ne rgy (in the form of ATP ) a nd inte rme dia te s for othe r me ta bolic pa thwa ys . Glycolys is is a t the hub of ca rbohydra te me ta bolis m be ca us e virtua lly a ll s uga rs , whe the r a ris ing from the die t or from ca ta bolic re a ctions in the body, ca n ultima te ly be conve rte d to glucos e (Figure 8.9A). Pyruva te is the e nd product of glycolys is in ce lls with mitochondria a nd a n a de qua te s upply of oxyge n. This s e rie s of te n re a ctions is ca lle d a e robic glycolys is be ca us e oxyge n is re quire d to re oxidize the NADH forme d during the oxida tion of glyce ralde hyde 3-phos pha te (Figure 8.9B). Ae robic glycolys is s e ts the s ta ge for the oxida tive de ca rboxyla tion of pyruva te to a ce tyl CoA, a ma jor fue l of the TCA cycle . Alte rna tive ly, pyruva te is re duce d to la cta te a s NADH is oxidize d to NAD+ (Figure 8.9C). This conve rs ion of glucos e to la cta te is ca lle d a na e robic glycolys is be ca us e it ca n occur without the pa rticipa tion of oxyge n. Ana e robic glycolys is a llows the production of ATP in tis s ue s tha t la ck mitochondria (for e xa mple , re d blood ce lls a nd pa rts of the e ye ) or in ce lls de prive d of s ufficie nt oxyge n.

C

R R

+ C

P

C Active ca ta lytic unit of prote in kina se

Pro te in s ubs trate

ATP

ADP

Pho s pho rylate d pro te in H2 O P rote in phos pha ta s e

Pi

INTRACELLULAR EFFECTS

De pho s pho rylate d pro te in

Fig ure 8.8 Actions of cyclic AMP (cAMP ). P i = inorga nic phos pha te .

A

6-P Glucona te Ribulos e 5-P

Glycoge n

Ribos e 5-P

B

Ga la ctos e 1-P

Glucos e 1-P

Xylulos e 5-P

Glucos e 6-P

S e dohe ptulos e 7-P Erythros e 4-P

Fructos e 6-P

UDP -Ga la ctos e

Ae ro bic g lyc o lys is Gluc o s e 6-P

Glucos e

C Gluc o s e

Anae ro bic g lyc o lys is

Gluc o s e 6-P

Gluc o s e

Fructos e

Fructos e 1,6-bis -P Glyce ra lde hyde 3-P

Glucos e ca nnot diffus e dire ctly into ce lls but e nters by one of two tra ns port me cha nis ms : a Na +-inde pe nde nt, fa cilita te d diffus ion tra ns port s ys te m or a n ATP-de pe nde nt Na +-monos a ccha ride cotra ns port s ys te m.

Ga la ctos e

UDP -Glucos e

6-P Gluconola ctone

IV. TRANS PORT OF GLUCOS E INTO CELLS

Glyce ra lde hyde 3-P

Glyce ra lde hyde

Dihydroxya ce tone -P Glyce rol-P

1,3-Bis phos phoglyce ra te

Fruc to s e 6-P

Fruc to s e 6-P

Fruc to s e 1,6-bis pho s phate

Fruc to s e 1,6-bis pho s phate

Fructos e 1-P

Glyce rol

3-P hos phoglyce ra te Tria cylglyce rol

+

2-P hos phoglyce ra te

Ala Cys Gly Ser Thr Try NH3

Fa tty a cyl CoA

Fa tty a cid

P hos phoe nolpyruva te La cta te CO 2

CO 2

P yruva te CO 2

Ma lonyl CoA

Ace tyl-CoA

Ace toa ce ta te

As n Ca rba moyl-P

β-Hydroxybutyra te As pa rta te

Citrulline

Ornithine

Le u P he Tyr Trp Lys

Oxa loa ce ta te

Citra te

Ma la te

Is ocitra te CO 2 α-Ke togluta ra te

Argininos uccina te Fuma ra te Arginine

S uccina te

CO 2 S uccinyl CoA

Gln Glu

P ro His Arg

Me thylma lonyl CoA

Ure a

P he Tyr

Ile Me t Va l Thr

P ropionyl-CoA Ace tyl CoA Fa tty a cyl-CoA (odd-numbe r ca rbons )

Glyce ralde hyde 3-P

NAD

Dihydro xyac e to ne -P

+

Glyc e ralde hyde 3-P

NAD

NADH 1,3-Bis pho s pho g lyc e rate

NADH 1,3-Bis pho s pho g lyc e rate 3-Pho s pho g lyc e rate

3-Pho s pho g lyc e rate

2-Pho s pho g lyc e rate

2-Pho s pho g lyc e rate

Pho s pho e no lpyruvate

Pho s pho e no lpyruvate

Oxida tive phos phoryla tion

Pyruvate

Dihydro xyac e to ne -P

Lac tate

Pyruvate

Fig ure 8.9 A. Glycolys is s hown a s one of the e s s e ntia l pa thwa ys of e ne rgy me ta bolis m. B. Re a ctions of a e robic glycolys is . C. Re a ctions of a na e robic glycolys is . NAD(H) = nicotina mide a de nine dinucle otide ; P = phos pha te .

tahir99-VRG & vip.persianss.ir

V. Re a ctions of Glycolys is A. S o dium-inde pe nde nt fac ilitate d diffus io n trans po rt s ys te m This s ys te m is me dia te d by a fa mily of 14 glucos e tra ns porte rs found in ce ll me mbra ne s . The y a re de s igna te d GLUT-1 to GLUT14 (glucos e tra ns porte r is oforms 1–14). The s e monome ric prote in tra ns porte rs e xis t in the me mbra ne in two conforma tiona l s ta te s (Figure 8.10). Extra ce llula r glucos e binds to the tra ns porte r, which the n a lte rs its conforma tion, tra ns porting glucos e a cros s the ce ll me mbra ne . 1. Tis s ue s pe c ific ity o f g luc o s e trans po rte r g e ne e xpre s s io n: The

GLUTs dis pla y a tis s ue -s pe cific pa tte rn of e xpre s s ion. For e xa mple , GLUT-3 is the prima ry glucos e tra ns porte r in ne urons . GLUT-1 is a bunda nt in e rythrocyte s a nd the blood–bra in ba rrie r but is low in a dult mus cle , whe re a s GLUT-4 is a bunda nt in mus cle a nd a dipos e tis s ue . [Note : The numbe r of GLUT-4 tra ns porte rs a ctive in the s e tis s ue s is incre a s e d by ins ulin. (Se e p. 311 for a dis cus s ion of ins ulin a nd glucos e tra ns port.)] GLUT-2 is a bunda nt in live r, kidne y, a nd β ce lls of the pa ncre a s . The othe r GLUT is oforms a lso ha ve tis s ue -s pe cific dis tributions . 2. S pe c ialize d func tio ns o f g luc o s e trans po rte r is o fo rms : In fa cili-

ta te d diffus ion, tra ns porte r-me dia te d glucos e move me nt is down a conce ntra tion gra die nt (tha t is , from a high glucos e conce ntra tion to a lowe r one a nd, the re fore , doe s not re quire e nergy). For e xa mple , GLUT-1, GLUT-3, a nd GLUT-4 a re prima rily involve d in glucos e upta ke from the blood. In contra s t, GLUT-2, in the live r and kidne y, ca n e ithe r tra ns port glucos e into the s e ce lls whe n blood glucos e le ve ls a re high or tra ns port glucos e from the s e cells whe n blood glucos e le ve ls a re low (for e xa mple , during fa s ting). GLUT-5 is unus ua l in tha t it is the prima ry tra ns porte r for fructos e (not glucos e ) in the s ma ll inte s tine a nd the te s te s . B. S o dium–mo no s ac c haride c o trans po rt s ys te m This is a n e ne rgy-re quiring proce s s tha t tra ns ports glucos e “a ga ins t” a conce ntra tion gra die nt (tha t is , from low glucos e conce ntra tions outs ide the ce ll to highe r conce ntra tions within the ce ll). This sys te m is a tra ns porte r-me dia te d proce s s in which the move me nt of glucos e is couple d to the conce ntra tion gra die nt of Na +, which is tra ns porte d into the ce ll a t the s a me time . The tra ns porte r is a s odium-de pe nde nt glucos e tra ns porte r (S GLT). This type of tra ns port occurs in the e pithe lia l ce lls of the inte s tine (s e e p. 87), re na l tubule s , a nd choroid ple xus . [Note : The choroid ple xus , pa rt of the blood–bra in ba rrie r, a ls o conta ins GLUT-1.]

V. REACTIONS OF GLYCOLYS IS The conve rs ion of glucos e to pyruva te occurs in two s ta ge s (Figure 8.11). The firs t five re a ctions of glycolys is corre s pond to a n e ne rgy– inve s tme nt pha s e in which the phos phoryla te d forms of inte rme dia te s a re s ynthe s ize d a t the e xpe ns e of ATP . The s ubs e que nt re a ctions of glycolys is cons titute a n e ne rgy–ge ne ra tion pha s e in which a ne t of two mole cule s of ATP a re forme d by s ubs tra te -le ve l phos phoryla tion (s e e p. 102) pe r glucos e mole cule me ta bolize d.

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Gluc o s e Gluc o s e trans po rte r (s tate 1)

Extra ce llula r s pa ce

Ce ll me mbra ne GLUT Cytos ol

Gluc o s e trans po rte r (s tate 2)

Extra ce llula r s pa ce

Cytos ol

Fig ure 8.10 S che ma tic re pre s e nta tion of the fa cilita te d tra ns port of glucos e through a ce ll me mbra ne . [Note : Glucos e tra ns porte r prote ins a re monome ric a nd conta in 12 tra ns me mbra ne α he lice s .]

Gluc o s e

Ene rg yinve s tme nt phas e

Ene rg yg e ne ratio n phas e

2ADP

4ADP

2NAD+

2ATP

4ATP

2NADH

2Pyruvate Ne t (ae ro bic g lyc o lys is ): Gluc o s e 2ADP 2NAD+

2Pyruvate 2ATP 2NADH

Fig ure 8.11 Two pha s e s of a e robic glycolys is . NAD(H) = nicotina mide a de nine dinucle otide .

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8. Introduction to Me ta bolis m a nd Glycolys is A. Pho s pho rylatio n o f g luc o s e

O C H H C OH HO C H H C OH H C OH H C OH H D-Gluc o s e ATP He xokina s e Glucokina s e

ADP O C H H C OH HO C H H C OH H C OH H C O P H Gluc o s e 6-pho s phate

Fig ure 8.12 Ene rgy-inve s tme nt pha s e : phos phoryla tion of glucos e . [Note : Kina s e s utilize ATP comple xe d with a diva le nt me ta l ion, mos t typica lly Mg 2+.]

Phos phoryla te d s uga r mole cule s do not re a dily pe ne tra te ce ll me mbra nes because the re are no spe cific transmembrane carriers for the se compounds a nd be ca us e the y a re too pola r to diffus e through the lipid core of me mbra ne s. The irre versible phosphoryla tion of glucose (Figure 8.12), therefore, effective ly traps the suga r as cytosolic glucose 6-phos pha te , the re by committing it to furthe r me ta bolis m in the ce ll. Ma mma ls ha ve four (I–IV) is ozyme s of the e nzyme he xokina s e tha t cata lyze the phosphorylation of glucose to glucose 6-phosphate. 1. He xo kinas e s I–III: In mos t tis s ue s , phos phoryla tion of glucos e is

ca ta lyze d by one of the s e is ozyme s of he xokina s e , which is one of thre e re gula tory e nzyme s of glycolys is (s e e a ls o phos phofructokina s e a nd pyruva te kina s e). The s e is ozyme s ha ve broa d s ubs tra te s pe cificity a nd a re a ble to phos phoryla te s e ve ra l he xos e s in a ddition to glucose . The y a re inhibite d by the rea ction product, glucose 6-phosphate, which accumulates when further metabolism of this hexose phosphate is re duced. Hexokinases I-III have a low Michaelis consta nt (Km ) (a nd, therefore , a high affinity; see p. 59) for glucose . This permits the efficient phosphorylation a nd subsequent metabolism of glucose eve n when tissue concentrations of glucose a re low (Figure 8.13). These isozymes, howeve r, ha ve a low maxima l ve locity ([Vma x] s e e p. 59) for glucos e a nd, the re fore , do not s e que s te r (tra p) ce llula r phos pha te in the form of phos phoryla te d hexoses, or phosphorylate more sugars tha n the cell can use. 2. He xo kinas e IV (o r, g luc o kinas e ): In live r pa re nchyma l ce lls a nd

Co nc e ntratio n o f fas ting blo o d g luc o s e

Enzyme ac tivity

Vma x Glucokina s e

Glu c o kin a s e

Vma x He xokina s e

He xo kin a s e 0

0

Km He xokina s e

5

10

15

20

Km Glucokina s e

Gluc o s e c o nc e ntratio n (mmo l/l)

Fig ure 8.13 Effe ct of glucos e conce ntra tion on the ra te of phos phoryla tion ca ta lyze d by he xokina s e a nd glucokina s e . Km = Micha e lis cons ta nt; Vma x = ma xima l ve locity.

β ce lls of the pa ncre a s , glucokina s e (the he xokina s e IV is ozyme ) is the pre domina nt e nzyme re s pons ible for the phos phoryla tion of glucos e . In β ce lls , glucokina s e functions a s a glucos e s e ns or, de te rmining the thre s hold for ins ulin s e cre tion (s e e p. 309). [Note : He xokina s e IV a ls o s e rve s a s a glucos e s e ns or in ne urons of the hypotha la mus , pla ying a ke y role in the a dre ne rgic re s pons e to hypoglyce mia (s e e p. 315.] In the live r, the e nzyme fa cilita te s glucos e phos phoryla tion during hype rglyce mia . De s pite the popula r but mis le a ding na me glucokina s e , the s uga r s pe cificity of the e nzyme is s imila r to tha t of othe r he xokina s e is ozyme s . a. Kine tic s : Glucokina s e diffe rs from he xokina s e s I–III in s e ve ra l importa nt prope rtie s . For e xa mple , it ha s a much highe r Km , re quiring a highe r glucos e conce ntra tion for ha lf-s a tura tion (s e e Figure 8.13). Thus , glucokina s e functions only whe n the intra ce llula r conce ntra tion of glucos e in the he pa tocyte is e le va te d s uch a s during the brie f pe riod following cons umption of a ca rbohydra te -rich me a l, whe n high le ve ls of glucos e a re de live re d to the live r via the porta l ve in. Glucokina s e ha s a high Vma x, a llowing the live r to e ffe ctive ly re move the flood of glucos e de live re d by the porta l blood. This pre ve nts la rge a mounts of glucos e from e nte ring the s ys te mic circula tion following s uch a me a l the re by minimizing hype rglyce mia during the a bs orptive pe riod. [Note : GLUT-2 ins ure s tha t blood glucos e e quilibra te s ra pidly a cros s the me mbra ne of the he pa tocyte .] b. Regulation by fructos e 6-pho s phate and glucos e: Glucokinase activity is not directly inhibite d by glucose 6-phosphate as are the other hexokinase s but, rather, is indire ctly inhibited by fructose 6-phosphate (which is in equilibrium with glucose

tahir99-VRG & vip.persianss.ir

V. Re a ctions of Glycolys is Gluc o s e GLUT-2

CYTOS OL

Gluc o s e GK

+

Gluc o s e 6-pho s phate

NUCLEUS GK

Fruc to s e 6-pho s phate

Pyruvate

Glucokina s e functions a s a glucos e s e ns or in the ma inte na nce of blood glucos e home os ta s is . Ina ctiva ting muta tions of glucokina s e a re the ca us e of a ra re form of dia be te s , ma turity ons e t dia be te s of the young type 2 (MODY 2) tha t is cha ra cte rize d by impa ire d ins ulin s e cre tion.

PLAS MA MEMBRANE

+

6-phospha te , a product of glucokina se) and is indire ctly s timula te d by glucos e (a s ubs tra te of glucokina s e) via the following mecha nism. Glucokina se re gula tory protein (GKRP) in the liver regulates the activity of glucokinase through reversible binding. In the presence of fructose 6-phosphate, glucokinase is transloca ted into the nucleus and binds tightly to the re gula tory protein, thereby rendering the enzyme ina ctive (Figure 8.14). When glucose levels in the blood (a nd also in the hepa tocyte, as a result of GLUT-2) increase, glucokinase is relea sed from the regulatory protein, and the enzyme reenters the cytosol whe re it phosphorylates glucose to glucose 6-phosphate. [Note : Fructose 1-phosphate inhibits forma tion of the glucokina se–GKRP complex.]

99

GKRP

Gluc o kinas e re g ulato ry Glu c o pro te in kin a s e (GK) (GKRP) (inac tive )

Fig ure 8.14 Re gula tion of glucokina s e a ctivity by glucokina s e re gula tory prote in. GLUT = glucos e tra ns porte r.

B. Is o me rizatio n o f g luc o s e 6-pho s phate The is ome riza tion of glucos e 6-phos pha te to fructos e 6-phos pha te is ca ta lyze d by phos phoglucos e is ome ra s e (Figure 8.15). The re a ction is re a dily re ve rs ible a nd is not a ra te -limiting or re gula te d s te p. C. Pho s pho rylatio n o f fruc to s e 6-pho s phate The irre ve rs ible phos phoryla tion re a ction ca ta lyze d by phos phofructokina s e -1 (P FK-1) is the mos t importa nt control point a nd the ra te -limiting a nd committe d s te p of glycolys is (Figure 8.16). P FK-1 is controlle d by the a va ila ble conce ntra tions of the s ubs tra te s ATP a nd fructos e 6-phos pha te a s we ll a s by re gula tory s ubs ta nce s de s cribe d be low. 1. Re g ulatio n by e ne rg y le ve ls within the c e ll: P FK-1 is inhibite d

a llos te rica lly by e le va te d le ve ls of ATP , which a ct a s a n “e ne rgyrich” s igna l indica ting a n a bunda nce of high-e ne rgy compounds . Ele va te d le ve ls of citra te , a n inte rme dia te in the TCA cycle (s e e p. 109), a ls o inhibit P FK-1. [Note : Inhibition by citra te fa vors the us e of glucos e for glycoge n s ynthe s is (s e e p. 125).] Conve rs e ly, P FK-1 is a ctiva te d a llos te rica lly by high conce ntra tions of AMP , which s igna l tha t the ce ll’s e ne rgy s tore s a re de ple te d. 2. Re g ulatio n by fruc to s e 2,6-bis pho s phate : Fructos e 2,6-bis phos -

pha te is the mos t pote nt a ctiva tor of P FK-1 (s e e Figure 8.16) a nd is a ble to a ctiva te the e nzyme e ve n whe n ATP le ve ls a re high. Fructos e 2,6-bis phos pha te is forme d from fructos e 6-phos pha te by phos phofructokina s e -2 (P FK-2), a n e nzyme diffe re nt tha n P FK-1. P FK-2 is a bifunctiona l prote in tha t ha s both the kina s e a ctivity tha t produce s fructos e 2,6-bis phos pha te a nd the phos pha ta s e a ctivity tha t de phos phoryla te s fructos e 2,6-bis phos pha te ba ck to fructos e 6-phos pha te . In the live r, the kina s e doma in is a ctive if de phos phoryla te d a nd is ina ctive if phos phoryla te d (Figure 8.17). [Note : Fructos e 2,6-bis phos pha te is a n inhibitor

O C H H C OH HO C H H C OH H C OH H C O P H Gluc o s e 6-pho s phate (a ldos e ) P hos phoglucos e is ome ra s e

H HO H H H

H C C C C C C H

OH

O H OH OH O P

Fruc to s e 6-pho s phate (ke tos e )

Fig ure 8.15 Aldos e -ke tos e is ome riza tion of glucos e 6-phos pha te to fructos e 6-phos pha te . P = phos pha te .

tahir99-VRG & vip.persianss.ir

100

8. Introduction to Me ta bolis m a nd Glycolys is of fructos e 1,6-bis phos pha ta s e , a n e nzyme of glucone oge ne s is (s e e p. 120). The re ciproca l a ctions of fructos e 2,6-bis phos pha te on glycolys is (a ctiva tion) a nd glucone oge ne s is (inhibition) e ns ure tha t both pa thwa ys a re not fully a ctive a t the s a me time , pre ve nting a futile cycle in which glucos e would be conve rte d to pyruva te followe d by re s ynthe s is of glucos e from pyruva te .]

Fruc to s e 6-pho s phate ATP, c itrate

ATP

AMP Fruc to s e 2,6-bis pho s phate

+ +

P hos phofructokina s e -1

ADP H H C O P C O HO C H

a. During the we ll-fe d s tate : De cre a s e d le ve ls of gluca gon a nd e le va te d le ve ls of ins ulin, s uch a s occur following a ca rbohydra te -rich me a l, ca us e a n incre a s e in fructos e 2,6-bis phos pha te a nd, thus , in the ra te of glycolys is in the live r (s e e Figure 8.17). Fructos e 2,6-bis phos pha te , the re fore , a cts a s a n intra ce llula r s igna l, indica ting tha t glucos e is a bunda nt.

H C OH H C OH H C O P H

Fruc to s e 1,6-bis pho s phate Aldola s e

O C H H C OH H C O P H

Trios e phos pha te is omera s e

Glyc e ralde hyde 3-pho s phate

H H C O P C O H C OH H

b. During fas ting : Ele va te d le ve ls of gluca gon a nd low le ve ls of ins ulin, s uch a s occur during fa s ting (s e e p. 327), de cre a s e the intra ce llula r conce ntra tion of he pa tic fructos e 2,6-bis phos pha te . This re s ults in inhibition of glycolys is a nd a ctiva tion of glucone oge ne s is .

Dihydro xyac e to ne pho s phate

D. Cle avag e o f fruc to s e 1,6-bis pho s phate Fig ure 8.16 Ene rgy–inve s tme nt pha s e (continue d): Conve rs ion of fructos e 6-phos pha te to trios e phos pha te s . P = phos pha te ; AMP = a de nos ine monophos pha te .

Aldola s e cle a ve s fructos e 1,6-bis phos pha te to dihydroxya ce tone phos pha te a nd glyce ra lde hyde 3-phos pha te (s e e Figure 8.16). The re a ction is re ve rs ible a nd not re gula te d. [Note : Aldola s e B, the is oform found prima rily in the live r, a ls o cle a ve s fructos e 1-phos pha te a nd functions in the me ta bolis m of die ta ry fructos e (s e e p. 138).]

Ins ulin (hig h)

Gluc ag o n (lo w) Re ce ptor

CELL MEMBRANE

Ad e n ylyl c yc la s e ATP c AMP

CYTOS OL

Re ce ptor Ac tivatio n o f many e nzyme s Hig h ins ulin /g luc ag o n ratio c aus e s de c re as e d c AMP and re duc e d le ve ls o f ac tive p ro te in kin a s e A.

1 G ly c o ly s is Glucos e 6-P

F r u c t o s e 6 -p h o s p h a t e

Glucos e

Fr u c t o s e 6 -P

Glyce ra lde hyde 3-P

DHAP

2

Fruc to s e 6-pho s phate

De c re as e d p ro te in kin a s e A ac tivity favo rs de pho s pho rylatio n o f P FK-2/FBP -2.

AT P P h o s p h o fru c to kin a s e -1

+

F r u c t o s e 1 ,6 -b is -P

Ac tive p ro te in kin a s e A

ADP

P

Bifunctiona l e nzyme P FK-2 (a ctive )

ATP

ADP

FBP -2 (ina ctive )

PFK-2 (inactive)

FBP -2 (a ctive )

1,3-Bis phos phoglyce ra te 3-P hos phoglyce ra te

F r u c t o s e 1 ,6 -b is p h o s p h a t e

P Fruc to s e

Bifunctiona l e nzyme

2,6-bis pho s phate

2-P hos phoglyce ra te P hos phoe nolpyruva te

Ele vate d c o nc e ntratio n o f fruc to s e 2,6-bis pho s phate ac tivate s P FK-1, whic h le ads to an inc re as e d rate o f g lyc o lys is .

La cta te

4

P yruva te

3

De pho s pho rylate d P FK-2 do main is ac tive , whe re as FBP -2 is inac tive whic h favo rs fo rmatio n o f fruc to s e 2,6-bis pho s phate .

Fig ure 8.17 Effe ct of e le va te d ins ulin conce ntra tion on the intra ce llula r conce ntra tion of fructos e 2,6-bis phos pha te in live r. P FK-2 = phos phofructokina s e -2; FBP -2 = fructos e 2,6-bis phos pha ta s e ; cAMP = cyclic AMP ; P = phos pha te .

tahir99-VRG & vip.persianss.ir

V. Re a ctions of Glycolys is E. Is o me rizatio n o f dihydro xyac e to ne pho s phate Trios e phos pha te is ome ra s e inte rconve rts dihydroxya ce tone phos pha te (DHAP ) a nd glyce ra lde hyde 3-phos pha te (s e e Figure 8.16). DHAP mus t be is ome rize d to glyce ra lde hyde 3-phos pha te for furthe r me ta bolis m by the glycolytic pa thwa y. This is ome riza tion re s ults in the ne t production of two mole cule s of glyce ra lde hyde 3-phos pha te from the cle a va ge products of fructos e 1,6-bis phos pha te . [Note : DHAP is utilize d in tria cylglyce rol s ynthe s is (s e e p. 188).] F. Oxidatio n o f g lyc e ralde hyde 3-pho s phate The conve rs ion of glyce ra lde hyde 3-phos pha te to 1,3-bis phos phoglyce ra te (1,3-BP G) by glyce ra lde hyde 3-phos pha te de hydroge na s e is the firs t oxida tion-re duction re a ction of glycolys is (Figure 8.18). [Note : Be ca us e the re is only a limite d a mount of NAD+ in the ce ll, the NADH forme d by this re a ction mus t be re oxidize d to NAD+ for glycolys is to continue . Two ma jor me cha nis ms for oxidizing NADH a re 1) the NADH-linke d conve rs ion of pyruva te to la cta te (a na e robic; s e e p. 96) a nd 2) oxida tion of NADH via the re s pira tory cha in (a e robic; s e e p. 74). The la tte r re quire s the ma la te -a s pa rta te a nd glyce rol 3-phos pha te s ubs tra te s huttle s (s e e p. 79.] 1. Synthe s is o f 1,3-bis pho s pho g lyc e rate : The oxida tion of the a lde -

hyde group of glyce ra lde hyde 3-phos pha te to a ca rboxyl group is couple d to the a tta chme nt of P i to the ca rboxyl group. The highe ne rgy phos pha te group a t ca rbon 1 of 1,3-BPG cons e rve s much of the fre e e ne rgy produce d by the oxida tion of glyce ra lde hyde 3-phos pha te . The e ne rgy of this high-e ne rgy phos pha te drive s the s ynthe s is of ATP in the ne xt re a ction of glycolys is. 2. Me c hanis m o f ars e nic po is o ning : The toxicity of a rs e nic is

3. S ynthe s is o f 2,3-bis pho s pho g lyc e rate in re d blo o d c e lls : S ome of the 1,3-BP G is conve rte d to 2,3-BP G by the a ction of bis phos phoglyce ra te muta s e (s e e Figure 8.18). 2,3-BP G, which is found in only tra ce a mounts in mos t ce lls , is pre s e nt a t high conce ntra tion in re d blood ce lls (RBCs ) a nd s e rve s to incre a s e O 2 de live ry (s e e p. 31). 2,3-BP G is hydrolyze d by a phos pha ta s e to 3-phos phoglyce ra te , which is a ls o a n inte rme dia te in glycolys is (s e e Figure 8.18). In the RBC, glycolys is is modifie d by inclus ion of the s e “s hunt” re a ctions .

O C H H C OH H C O P H Glyc e ralde hyde 3-pho s phate Pi NAD+

Glyce ra lde hyde 3-phos pha te de hydroge na s e

NADH + H+

O C O~ P H C OH H C O P H 1,3-Bis pho s pho g lyc e rate Muta s e O

ADP P hos phoglyce ra te kina s e

ATP O C OH C OH H C O P H

C OH C O P H C O P H 2,3-Bis pho s pho g lyc e rate H2 O P hos pha ta s e

Pi

3-Pho s pho g lyc e rate P hos phoglyce ra te muta s e

O C OH C O P H C OH H 2-Pho s pho g lyc e rate Enola s e

H2 O

O C OC O~ P H C H Pho s pho e no lpyruvate ADP P yruva te kina s e

Fruc to s e 1,6bis pho s phate

+

due prima rily to the inhibition by triva le nt a rs e nic (a rs e nite ) of e nzyme s s uch a s the pyruva te de hydroge na s e comple x, which re quire lipoic a cid a s a coe nzyme (s e e p. 110). Howe ve r, pe nta va le nt a rs e nic (a rs e na te ) ca n pre ve nt ne t ATP a nd NADH production by glycolys is without inhibiting the pa thwa y its e lf. It doe s s o by compe ting with P i a s a s ubs tra te for glyce ra lde hyde 3-phos pha te de hydroge na s e , forming a comple x tha t s ponta ne ous ly hydrolyze s to form 3-phos phoglyce ra te (s e e Figure 8.18). By bypa s s ing the s ynthe s is of a nd phos pha te tra ns fe r from 1,3BP G, the ce ll is de prive d of e ne rgy us ua lly obta ine d from the glycolytic pa thwa y. [Note : Ars e na te a ls o compe te s with P i on the F 1 doma in of ATP s yntha s e (s e e p. 77), re s ulting in forma tion of ADP -a rs e na te tha t is ra pidly hydrolyze d.]

101

O C OC O H C H H Pyruvate

ATP

Fig ure 8.18 Ene rgy–ge ne ra ting pha s e : conve rs ion of glyce ra lde hyde 3-phos pha te to pyruva te . NAD(H) = nicotina mide a de nine dinucle otide P = phos pha te ; P i = inorga nic phos pha te .

102

8. Introduction to Me ta bolis m a nd Glycolys is G. S ynthe s is o f 3-pho s pho g lyc e rate , pro duc ing ATP Whe n 1,3-BP G is conve rte d to 3-phos phoglyce ra te , the high-e ne rgy phos pha te group of 1,3-BP G is us e d to s ynthe s ize ATP from ADP (s e e Figure 8.18). This re a ction is ca ta lyze d by phos phoglyce ra te kina s e , which, unlike mos t othe r kina s e s , is phys iologica lly re ve rs ible . Be ca us e two mole cule s of 1,3-BPG a re forme d from e a ch glucos e mole cule , this kina s e re a ction re pla ce s the two ATP mole cule s cons ume d by the e a rlie r forma tion of glucos e 6-phospha te a nd fructos e 1,6-bis phos pha te . [Note : This is a n e xa mple of s ubs tra te -le ve l phos phoryla tion, in which the e ne rgy ne e de d for the production of a high-e ne rgy phos pha te come s from a s ubs tra te ra the r tha n from the e le ctron tra ns port cha in (s e e J . be low a nd p. 113 for othe r e xa mple s ).] H. Shift of the phos phate group The s hift of the phos pha te group from ca rbon 3 to ca rbon 2 of phos phoglyce ra te by phos phoglyce ra te muta s e is fre e ly re ve rs ible (s e e Figure 8.18). I. De hydratio n o f 2-pho s pho g lyc e rate The de hydra tion of 2-phos phoglyce ra te by e nola s e re dis tribute s the energy within the substrate, resulting in the formation of phosphoenolpyruva te (P EP ), which conta ins a high-e ne rgy e nol phos pha te (s e e Figure 8.18). The reaction is reversible despite the high-energy nature of the product. [Note: Fluoride inhibits enolase, and water fluoridation reduces lactate production by mouth bacteria, decreasing dental caries.] J. Fo rmatio n o f pyruvate , pro duc ing ATP

Re ce ptor

The conve rs ion of P EP to pyruva te is ca ta lyze d by pyruva te kina s e (P K), the third irre ve rs ible re a ction of glycolys is . The high-e ne rgy e nol phos pha te in PEP is us e d to s ynthe s ize ATP from ADP a nd is a nothe r e xa mple of s ubs tra te -le ve l phos phoryla tion (s e e Figure 8.18).

Ade nylyl cycla s e

1. Fe e dfo rward re g ulatio n: P K is a ctiva te d by fructos e 1,6-bis pho-

Gluc ag o n

ATP

c AMP + PP i

Ac tive p ro te in kin a s e A PEP ADP

2. Co vale nt mo dulatio n o f pyruvate kinas e : P hos phoryla tion by P

ATP

ADP

P yruva te kina s e (a ctive )

P yruva te kina s e (ina ctive )

ATP

s pha te , the product of the phos phofructokina s e -1 re a ction. This fe e dforwa rd (ins te a d of the more us ua l fe e dba ck) re gula tion ha s the e ffe ct of linking the two kina s e a ctivitie s : incre a s e d phos phofructokina s e a ctivity re s ults in e le va te d le ve ls of fructos e 1,6-bis phos pha te , which a ctiva te s P K.

Pyruvate

Fig ure 8.19 Cova le nt modifica tion of he pa tic pyruvate kinase results in inactivation of the e nzyme . cAMP = cyclic AMP ; P EP = phos phoe nolpyruva te ; P = phos pha te ; P P i = pyrophos pha te .

a cAMP -de pe nde nt prote in kina s e le a ds to ina ctiva tion of the he pa tic is ozyme of P K (Figure 8.19). Whe n blood glucos e le ve ls a re low, e le va te d gluca gon incre a s e s the intra ce llula r le ve l of cAMP , which ca us e s the phos phoryla tion a nd ina ctiva tion of P K in the live r only. The re fore , P EP is una ble to continue in glycolys is a nd, ins te a d, e nte rs the glucone oge ne s is pa thwa y. This , in pa rt, e xpla ins the obs e rve d inhibition of he pa tic glycolys is a nd s timula tion of glucone oge ne s is by gluca gon. De phos phoryla tion of P K by a phos pha ta s e re s ults in re a ctiva tion of the e nzyme . 3. Pyruvate kinas e de fic ie nc y: Ma ture RBCs la ck mitochondria a nd

a re, therefore , completely dependent on glycolysis for ATP production. ATP is required to meet the metabolic needs of RBCs and to fue l the ion pumps nece ss a ry for the ma intena nce of the fle xible , biconca ve sha pe that a llows them to squeeze through narrow capil-

V. Re a ctions of Glycolys is laries. The anemia observed in glycolytic enzyme deficiencie s is a conseque nce of the reduce d rate of glycolysis, lea ding to decrease d ATP production. The re s ulting a lte ra tions in the RBC me mbra ne le a d to cha nge s in ce ll s ha pe a nd, ultima te ly, to pha gocytos is by ce lls of the reticuloendothelial system, pa rticularly macropha ges of the splee n. The premature dea th and lysis of RBCs result in hemolytic ane mia. Among patients exhibiting the rare genetic de fects of glycolytic enzymes, the majority has a de ficiency in PK. The effects of PK deficie ncy are restricted to RBCs and include mild-to-se vere nons phe rocytic he molytic a ne mia , with the s e ve re form re quiring re gula r tra ns fus ions . [Note : He pa tic P K is e ncode d by the s a me ge ne a s the RBC is ozyme . Live r ce lls s how no e ffe ct, howe ve r, be ca us e the y ha ve mitochondria a nd ca n ge ne ra te ATP by oxida tive phos phoryla tion.] S e ve rity de pe nds both on the de gre e of enzyme deficiency (genera lly 5–35% of normal levels) and on the extent to which RBCs compe nsate by synthesizing increased levels of 2,3-BP G (s e e p. 31). Almos t a ll individua ls with P K de ficie ncy ha ve a muta nt e nzyme tha t s hows a bnorma l prope rtie s such a s altere d kinetics (Figure 8.20). Individuals hete rozygous for PK deficiency have resistance to the most severe forms of malaria .

103

Glucos e 6-P

Glucos e

Theee6-P nzyme may s ho w Fructos

an abno rmal re s po ns e to the ac tivato r fruc to s e uctos e 1,6-bis -P Fructos 1,6-bis pho s phate . Glyc ce ra lde hyde 3-P Glyce

The e nzyme may s ho w an abno rmal Km o r Vmax -P hos ra te fo r s1,3-bis ubs trate s phoglyce or c o e nzyme s .

DihydroxyDihydrroxyne -P ton a ce tone

3-P hhos phoglyce ra te 2-P hhos phoglyce ra te

Pho s pho e no lpyruvatee ADP P yru vaa te kin a s e

Fruc to s e 11,66bis pho s phate

+

ATP A Pyruvate

The tis s ue -s pe cific e xpre s s ion of P K in RBCs a nd the live r is the re s ult of diffe re ntia l promote r utiliza tion in tra ns cription (s e e p. 422) of the ge ne tha t e ncode s both is ozyme s .

K. Re duc tio n o f pyruvate to lac tate La cta te , forme d by the a ction of la cta te de hydroge na s e , is the fina l product of a na e robic glycolys is in e uka ryotic ce lls (Figure 8.21). The forma tion of la cta te is the ma jor fa te for pyruva te in the le ns a nd corne a of the e ye , kidne y me dulla , te s te s , le ukocyte s , a nd RBCs , be ca us e the s e a re a ll poorly va s cula rize d a nd/or la ck mitochondria .

La cta te

Enyme ac tivity o r s tability may be alte re d, o r the amo unt o f e nzyme may be de c re as e d.

Fig ure 8.20 Alte ra tions obs e rve d with va rious muta nt forms of pyruva te kina s e . Km = Micha e lis cons ta nt; Vma x = ma xima l ve locity. COO C O CH3

1. Lac tate fo rmatio n in mus c le : In e xe rcis ing s ke le ta l mus cle ,

NADH production (by glyce ra lde hyde 3-phos pha te de hydroge na s e a nd by the thre e NAD+-linke d de hydroge na s e s of the TCA cycle ; s e e p. 112) e xce e ds the oxida tive ca pa city of the re s pira tory cha in. This re s ults in a n e le va te d NADH/NAD+ ra tio, fa voring re duction of pyruva te to la cta te . The re fore , during inte ns e e xe rcis e , la cta te a ccumula te s in mus cle , ca us ing a drop in the intra ce llula r pH, pote ntia lly re s ulting in cra mps . Much of this la cta te e ve ntua lly diffus e s into the bloods tre a m a nd ca n be us e d by the live r to ma ke glucos e (s e e p. 118).

Pyruvate NADH + H+

NADH + H+ La cta te de hydroge na s e

NAD+

NAD+ COO HO C H CH3 Lac tate

2. Lac tate utilizatio n: The dire ction of the la cta te de hydroge na s e

re a ction de pe nds on the re la tive intra ce llula r conce ntra tions of pyruva te a nd la cta te a nd on the ra tio of NADH/NAD+ in the ce ll. For e xa mple , in the live r a nd he a rt, the ra tio of NADH/NAD+ is lowe r tha n in e xe rcis ing mus cle . The s e tis s ue s oxidize la cta te (obta ine d from the blood) to pyruva te . In the live r, pyruva te is e ithe r conve rte d to glucos e by glucone oge ne s is or oxidize d in the TCA cycle . He a rt mus cle e xclus ive ly oxidize s la cta te to CO 2 a nd H2 O via the TCA cycle .

Fig ure 8.21 Inte rconve rs ion of pyruva te a nd la cta te . [Note : La cta te produce d in mus cle e nte rs the circula tion, is picke d up by live r through fa cilita te d diffus ion, a nd is oxidize d to pyruva te . P yruva te is us e d by live r to ma ke glucos e .] NAD(H) = nicotina mide a de nine dinucle otide .

104

8. Introduction to Me ta bolis m a nd Glycolys is 3. Lac tic ac ido s is : Ele va te d conce ntra tions of la cta te in the pla s ma ,

ATP c o ns umptio n

te rme d la ctic a cidos is (a type of me ta bolic a cidos is ), occur whe n the re is a colla pse of the circula tory s ys te m, s uch as in myoca rdia l infa rction, pulmona ry e mbolis m, a nd uncontrolle d he morrha ge , or whe n a n individua l is in s hock. The fa ilure to bring a de qua te a mounts of oxyge n to the tis s ue s re s ults in impa ire d oxida tive phos phoryla tion a nd de cre a s e d ATP s ynthe s is . To s urvive , the ce lls re ly on a na e robic glycolys is for ge ne ra ting ATP , producing la ctic a cid a s the e nd product. [Note : Production of e ve n me a ge r a mounts of ATP ma y be life -s a ving during the pe riod re quire d to re e s ta blis h a de qua te blood flow to the tis s ue s .] The e xce s s oxyge n re quire d to re cove r from a pe riod whe n the a va ila bility of oxyge n ha s be e n ina de qua te is te rme d the “oxyge n de bt.”

Glucos e AT P ADP

Glucos e 6-P

Fructos e 6-P AT P ADP

NADH pro duc tio n

Fructos e 1,6-bis -P

Glyce ra lde hyde 3-P

DHAP

Pi

+

2 N AD + 2 N ADH + 2 H

2 (1,3-Bis phos phoglyce ra te ) 2 ADP 2 AT P 2 (3-P hos phoglyce ra te )

The oxyge n de bt is ofte n re la te d to pa tie nt morbidity or morta lity. In ma ny clinica l s itua tions , me a s uring the blood le ve ls of la ctic a cid a llows the ra pid, e a rly de te ction of oxyge n de bt in pa tie nts a nd the monitoring of the ir re cove ry.

L. Ene rg y yie ld fro m g lyc o lys is De s pite the production of s ome ATP during glycolys is , the e nd product, pyruva te or la cta te , s till conta ins mos t of the e ne rgy origina lly conta ine d in glucos e . The TCA cycle is re quire d to re le a s e tha t e ne rgy comple te ly (s e e p. 109). 1. Anae ro bic g lyc o lys is : Two mole cule s of ATP a re ge ne ra te d for

ATP pro duc tio n

2 (2-P hos phoglyce ra te )

2 (P hos phoe nolpyruva te ) 2 ADP 2 AT P

2 (La cta te )

2 N AD +

2 (P yruva te )

2 N ADH + 2 H +

NADH c o ns umptio n Fig ure 8.22 S umma ry of a na e robic glycolys is . Re a ctions involving the production or cons umption of ATP or NADH a re indica te d. The thre e irre ve rs ible re a ctions of glycolys is a re s hown with thick a rrows . DHAP = dihydroxya ce tone phos pha te ; NAD(H) = nicotina mide a de nine dinucle otide ; P = phos pha te .

e a ch mole cule of glucos e conve rte d to two mole cule s of la cta te (Figure 8.22). The re is no ne t production or cons umption of NADH. 2. Ae ro bic g lyc o lys is : The dire ct cons umption a nd forma tion of ATP

is the s a me a s in a na e robic glycolys is (tha t is , a ne t ga in of two ATP pe r mole cule of glucos e ). Two mole cule s of NADH a re a ls o produce d pe r mole cule of glucos e . Ongoing a e robic glycolys is re quire s the oxida tion of mos t of this NADH by the e le ctron tra ns port cha in, producing a pproxima te ly thre e ATP for e a ch NADH mole cule e nte ring the cha in (s e e p. 77). [Note : NADH ca nnot cros s the inne r mitochondria l me mbra ne , a nd s ubs tra te s huttle s a re re quire d (s e e p. 79).]

VI. HORMONAL REGULATION OF GLYCOLYS IS The re gula tion of glycolys is by a llos te ric a ctiva tion or inhibition, or the cova le nt phos phoryla tion/de phos phoryla tion of ra te -limiting e nzyme s , is s hort-te rm (tha t is , the y influe nce glucos e cons umption ove r pe riods of minute s or hours ). S upe rimpos e d on the s e mome nt-to-mome nt e ffe cts a re s lowe r, a nd ofte n more profound, hormona l influe nce s on ge ne e xpre s s ion, or the a mount of e nzyme prote in s ynthe size d. The s e e ffe cts ca n re s ult in 10-fold to 20-fold incre a s e s in e nzyme a ctivity tha t typica lly

VIII. Cha pte r S umma ry

Gluc o s e +

Glucokina s e

Fruc to s e 6-P P hos phofructokina s e

A. Oxidative de c arbo xylatio n o f pyruvate

Dihydroxya ce tone -P

1,3-Bis phos phoglyce ra te 3-P hos phoglyce ra te 2-P hos phoglyce ra te

Pho s pho e no lpyruvate Ins ulin P yruva te Gluc ag o n kina s e +

Oxida tive de ca rboxyla tion of pyruva te by the pyruva te de hydroge na s e comple x is a n importa nt pa thwa y in tis s ue s with a high oxida tive ca pa city s uch a s ca rdia c mus cle (Figure 8.24). P yruva te de hydroge na s e irre ve rs ibly conve rts pyruva te , the e nd product of glycolys is , into a ce tyl CoA, a ma jor fue l for the TCA cycle (s e e p. 109) a nd the building block for fa tty a cid s ynthe s is (s e e p. 183).

Ins ulin Gluc ag o n

Fruc to s e 1,6-bis pho s phate Glyce ra lde hyde 3-P

VII. ALTERNATE FATES OF PYRUVATE

Ins ulin Gluc ag o n

Gluc o s e 6-P

+

occur ove r hours to da ys . Although the curre nt focus is on glycolys is , re ciproca l cha nge s occur in the ra te -limiting e nzyme s of glucone oge ne s is , which a re de s cribe d in Cha pte r 10 (s e e p. 117). Re gula r cons umption of me a ls rich in ca rbohydra te or a dminis tra tion of ins ulin initia te s a n incre a s e in the a mount of glucokina s e , phos phofructokina s e , a nd PK in the live r (Figure 8.23). The s e cha nge s re fle ct a n incre a s e in ge ne tra ns cription, re s ulting in incre a s e d e nzyme s ynthe s is . High a ctivity of the s e thre e e nzyme s fa vors the conve rs ion of glucos e to pyruva te , a cha ra cte ris tic of the a bs orptive s ta te (s e e p. 321). Conve rs e ly, ge ne tra ns cription a nd s ynthe s is of glucokina s e, phos phofructokina s e , a nd P K a re de cre a s e d whe n pla s ma gluca gon is high a nd ins ulin is low (for e xa mple , a s s e e n in fa s ting or dia be te s ).

105

Pyruvate B. Carbo xylatio n o f pyruvate to o xalo ac e tate Ca rboxyla tion of pyruva te to oxa loa ce ta te by pyruva te ca rboxyla s e is a biotin-de pe nde nt re a ction (s e e Figure 8.24). This re a ction is importa nt be ca us e it re ple nis he s the TCA cycle inte rme diate s a nd provide s s ubs tra te for glucone oge ne s is (s e e p. 118). C. Re duc tio n o f pyruvate to e thano l (mic ro o rg anis ms ) The conve rs ion of pyruva te to e tha nol occurs by the two re a ctions s umma rize d in Figure 8.24. The de ca rboxyla tion of pyruva te by pyruva te de ca rboxyla s e occurs in ye a s t a nd ce rta in othe r microorga nis ms but not in huma ns . The e nzyme re quire s thia mine pyrophos pha te a s a coe nzyme a nd ca ta lyze s a re a ction s imila r to tha t de s cribe d for pyruva te de hydroge na s e (s e e p. 110).

VIII. CHAPTER S UMMARY Mos t pa thwa ys ca n be cla s s ifie d a s e ithe r c atabo lic (de g rade comple x mole cule s to a fe w s imple products ) or anabo lic (s ynthe s ize comple x e nd products from s imple pre curs ors ). Catabo lic re ac tio ns a ls o c apture c he mic al e ne rg y in the form of ATP from the de gra da tion of e ne rgyrich mole cule s . Anabo lic re ac tio ns re quire e ne rg y , which is ge ne ra lly provide d by the hydrolys is of ATP. The ra te of a meta bolic pa thwa y ca n re s pond to re gulatory s ignals s uch a s allo s te ric ac tivato rs or inhibito rs tha t a ris e from within the c e ll. S igna ling be twe e n c e lls provide s for the inte gra tion of me ta bolis m. The mos t importa nt route of this communica tion is c he mic al s ig naling (for e xa mple , by ho rmo ne s or ne uro trans mitte rs ). S e c o nd me s s e ng e r mo le c ule s tra ns duce a che mica l s igna l (hormone or ne urotra ns mitte r) to a ppropria te intra ce llula r re s ponde rs .

La cta te

Fig ure 8.23 Effe ct of ins ulin a nd gluca gon on the s ynthe s is of ke y e nzyme s of glycolys is in live r. P = phos pha te .

106

8. Introduction to Me ta bolis m a nd Glycolys is

ETHANOL S YNTHES IS

• Oc c urs in ye as t and

s o me bac te ria (inc luding inte s tinal flo ra)

• Thiamine pyro pho s phate – de pe nde nt pathway

Et h a n o l NAD+ NADH + H+ Ac e talde hyde

NAD+

La c t a t e

NADH + H+

CO2 TPP

P YR U VAT E NAD+

CO2

CO2

NADH + H+

O x a lo a c e t a t e

Ac e t y l C o A

P YRUVATE DEHYDROGENAS E COMP LEX

• Inhibite d by ac e tyl Co A

• S o urc e o f ac e tyl Co A fo r TCA c yc le and fatty ac id s ynthe s is

• An irre ve rs ible re ac tio n

P YRUVATE CARBOXYLAS E

• Ac tivate d by ac e tyl Co A ple nis he s inte rme diate s • oRef the TCA c yc le • Pro vide s s ubs trate s fo r g luc o ne o g e ne s is

• An irre ve rs ible re ac tio n Fig ure 8.24 S umma ry of the me ta bolic fa te s of pyruva te . TP P = thia mine pyrophos pha te . TCA = trica rboxylic a cid; NAD(H) = nicotina mide a de nine dinucle otide ; CoA = coe nzyme A.

Ad e n ylyl c yc la s e is a ce ll me mbra ne e nzyme tha t s ynthe s ize s c yc lic AMP (c AMP) in re s pons e to che mica l s igna ls , s uch a s the hormone s gluc ag o n a nd e pine phrine . Following binding of a hormone to its c e lls urfac e re c e pto r, a GTP -de pe nde nt re gula tory prote in (G pro te in) is activa te d tha t, in turn, a ctiva te s a de n ylyl c yc la s e. The cAMP produce d activa te s a pro te in kin a s e, which phos phoryla te s a ca dre of e nzyme s , ca us ing the ir a ctiva tion or de a ctiva tion. P hos phoryla tion is re ve rs e d by pro te in p h o s p h a ta s e s . Ae ro bic g lyc o lys is , in which pyruvate is the end product, occurs in ce lls with mitochondria a nd a n a de qua te s upply of oxyge n (Figure 8.25). Anae ro bic glyc o lys is , in which lac tic ac id is the end product, occurs in ce lls tha t la ck mitochondria a nd in ce lls de prive d of s ufficie nt oxyge n. Glucos e is tra ns porte d a cros s me mbra ne s by one of 14 g luc o s e trans po rte r is o fo rms (GLUTs ). GLUT-1 is a bunda nt in e rythro c yte s a nd the brain, GLUT-4 (which is ins ulin de pe nde nt) is found in mus c le a nd adipos e tis s ue , a nd GLUT-2 is found in the live r, kidne y, a nd β c e lls of the pa ncre a s . The conve rs ion of glucos e to pyruva te (glyc o lys is ; Figure 8.25) occurs in two s ta ge s : a n e ne rgy–inve s tme nt phas e in which phos phoryla te d inte rme dia te s a re s ynthe s ize d at the e xpe ns e of ATP , a nd a n e ne rgy-g e ne ration phas e , in which ATP is produce d. In the e ne rgy-inve s tme nt pha s e , glucose is phos phoryla te d by h e xo kin a s e (found in mo s t tis s ue s ) or g lu c o kin a s e (a he xokina s e found in live r c e lls a nd the β c e lls of the pa ncre a s ). He xokina s e ha s a high affinity (low Km ) a nd a low Vmax for glucos e a nd is inhibite d by g luc o s e 6-phos phate . Gluc okina s e ha s a high Km a nd a high Vmax for glucos e . It is indire ctly inhibite d by fruc to s e 6-phos phate a nd ac tivate d by gluc o s e . The trans c ription of the ge ne for glucokina s e is e nhance d by ins ulin. Glucos e 6-phos pha te is is ome rize d to fructos e 6-phos phate , which is phos phoryla te d to fruc to s e 1,6-bis pho s phate by p h o s p h o fruc tokina s e -1 (PFK-1). This e nzyme is allos te ric ally inhibite d by ATP a nd c itrate a nd ac tivate d by AMP. Fruc tos e 2,6-bis pho s phate , whos e s ynthe s is by p h os p h o fru c to kina s e -2 (PFK-2 ) is ac tivate d by ins ulin, is the most pote nt a lloste ric a ctiva tor of PFK-1. A tota l of two ATP are us e d during this pha s e of glycolys is . Fructos e 1,6-bis phos pha te is cle a ve d to form two trios e s tha t a re furthe r me ta bolize d by the glycolytic pa thwa y, forming pyruva te . During the se re a ctions, fo ur ATP a nd two NADH are pro duc e d from ADP a nd NAD+. The fina l s te p in pyruva te s ynthe s is from phos phoe nolpyruva te is ca ta lyze d by p yru va te kin a s e (P K). This e nzyme is allo s te ric ally ac tivated by fruc to s e 1,6-bis pho s phate a nd hormonally ac tivate d by ins ulin a nd inhibite d in the live r by g luc ag o n via the c AMP pathway. P K de fic ie nc y a ccounts for the ma jority of a ll inhe rite d de fe cts in glycolytic e nzyme s . Effe cts a re re s tricte d to e rythroc yte s a nd pre s e nt a s mild to s e ve re c hro nic , nons phe ro c ytic he molytic ane mia. In anae ro bic g lyc o lys is , NADH is re oxidize d to NAD+ by the c o nve rs io n o f pyruvate to lac tate . This occurs in ce lls , s uch a s e rythroc yte s , tha t ha ve fe w or no mitochondria , a nd in tis s ue s , s uch a s e xe rc is ing mus c le, whe re production of NADH e xce e ds the oxida tive ca pa city of the re s pira tory cha in. Ele va te d conce ntra tions of la cta te in the pla s ma (lac tic ac idos is ) occur whe n the re is a c o llaps e of the c irc ulato ry s ys te m or whe n a n individua l is in s hoc k. Pyruva te ca n be 1) oxidative ly de c arbo xylate d by p yru va te d e h ydro g e n a s e, producing ac e tyl c o e nzyme A; 2) c arbo xylate d to o xalo ac e tate (a trica rboxylic a cid cycle inte rme dia te ) by p yru va te c a rb o xyla s e ; or 3) re duc e d by microorga nis ms to e thano l by p yru va te d e c a rbo xyla s e.

VIII. Cha pte r S umma ry

107

Me tabo lic c harac te ris tic s o f g lyc o lys is

Re g ulatio n o f g lyc o lys is

Glyc o lys is

We ll-fe d s tate

cons is ts of

All tis s ue s

occurs in

Cyto s o l

occurs in

Inge s tion of glucos e

Gluc o s e Re g ulate d s te ps

ATP ATP

Blood glucos e Re le a s e of ins ulin

He xo kin a s e produce s

NADH NADH to be re o xidize d to NAD+

NADH

NADH

P rote in phos pha ta s e a ctivity

P h o s p h o fru c to kin a s e -1 P yru va te kin a s e (P K)

re quire s

Fructos e 2,6-bis phos pha te produce s

ATP

ATP

ATP

ATP

ATP

note worthy be ca us e

Fas ting s tate

Pyruvate Pyruvate Blood glucos e

re quire s

Ae ro bic me tabo lis m

Re le a s e of gluca gon

ma y be followe d by

ma y be followe d by

Oxyg e n to re o xidize NADH to NAD+ by the e le c tro n trans po rt c hain

Anae ro bic me tabo lis m

doe s not re quire

cAMP Oxyg e n P rote in kina s e a ctivity

cons is ts of

Pyruvate

Pyruvate NADH NAD+

Ac e tyl Co A

Lac tate

followe d by

NADH NAD+

Ethano l, CO2

occurs in

occurs in

Re d blo o d c e lls Exe rc is ing mus c le Ano xic tis s ue s

Ye as t S o me o the r mic ro o rg anis ms

TCA c yc le

Fructos e 2,6-bis phos pha te

Pyruvate

oxidize s Ac e tyl Co A

ca n re s ult in

2 CO2

Mutatio n in g e ne fo r P K

Lac tic ac ido s is

le a ds to

P K de fic ie nc y dis e as e

le a ds to

ca us ing

He mo lytic ane mia

Alte re d fo lding

le a ds to

Alte re d primary s truc ture o f e nzyme

Tricarboxylic Acid Cycle and Pyruvate Dehydrogenas e Complex

Co nc e pt c o nne c t

Fig ure 8.25 Ke y conce pt ma p for glycolys is . NAD(H) = nicotina mide a de nine dinucle otide ; cAMP = cyclic a de nos ine monophos pha te ; CoA = coe nzyme A; TCA = trica rboxylic a cid.

9

108

8. Introduction to Me ta bolis m a nd Glycolys is

S tudy Que s tio ns Choos e the ONE be s t a ns we r. 8.1 Which of the following be s t de s cribe s the a ctivity le ve l a nd phos phoryla tion s ta te of the lis te d he pa tic e nzyme s in a n individua l who cons ume d a ca rbohydra te -rich me a l a bout a n hour a go? P FK-1 = phosphofructokina s e -1; P FK-2 = phos phofructokina s e -2; P = phosphoryla te d. Choice

P FK-1

P FK-2

P yruva te Kina s e

Activity

P

Activity

P

Activity

P

A.

Low

No

Low

No

Low

No

B.

High

Ye s

Low

Ye s

Low

Ye s

C.

High

No

High

No

High

No

D.

High

Ye s

High

Ye s

High

Ye s

8.2 Which of the following s ta te me nts is true for a na bolic pa thwa ys only? A. The ir irre ve rs ible (none quilibrium) re a ctions a re re gula te d. B. The y a re ca lle d cycle s if the y re ge ne ra te a n inte rme dia te . C. The y a re conve rge nt a nd ge ne ra te a fe w s imple products . D. The y a re s ynthe tic a nd re quire e ne rgy. E. The y typica lly re quire oxidize d coe nzyme s . 8.3 Compa re d with the re s ting s ta te , vigorous ly contra cting s ke le ta l mus cle s hows : A. B. C. D. E.

de cre a s e d AMP /ATP ra tio. de cre a s e d le ve ls of fructos e 2,6-bis phos pha te . de cre a s e d NADH/NAD+ ra tio. incre a s e d oxyge n a va ila bility. incre a s e d re duction of pyruva te to la cta te .

8.4 Glucos e upta ke by: A. live r ce lls is through fa cilita te d diffus ion involving a glucos e tra ns porte r. B. inte s tina l mucos a l ce lls re quire s ins ulin. C. b ra in c e lls is th ro ug h e n e rg y-re q u irin g (a c tive ) tra ns port. D. mos t ce lls is through s imple diffus ion up a conce ntra tion gra die nt. 8.5 Give n tha t the Km of glucokina s e for glucos e is 10 mM whe re a s tha t of he xokina s e is 0.1 mM, which is ozyme will more clos e ly a pproa ch Vma x a t the norma l blood glucos e conce ntra tion of 5 mM? 8.6 In pa tie nts with whooping cough, Gα i is inhibite d. How doe s this le a d to a ris e in cyclic AMP ?

Corre ct a ns we r = C. In the pe riod imme dia te ly following a me a l, blood gluc os e le ve ls a nd he pa tic upta ke of glucos e incre a se . The glucose is phos phoryla te d to glucos e 6-phos pha te a nd use d in glycolysis. In re s ponse to the ris e in blood glucos e , the ins ulin-to-gluca gon ra tio incre a s e s . As a re s u lt, th e kina s e do ma in of P FK-2 is de phos phoryla te d a nd a ctive . Its product, fructos e 2,6-bis phos pha te , a llos te rica lly a ctiva te s PFK-1. (PFK-1 is not covalently regulate d.) Active P FK-1 produce s fructos e 1,6-bis phos pha te tha t is a fe e dforwa rd a ctiva tor of pyruva te kina s e . He pa tic pyruva te kina s e is cova le ntly re gula te d, a nd the rise in insulin fa vors de phos phoryla tion.

Corre ct a ns we r = D. Ana bolic proce s s e s a re s ynthe tic a nd e ne rgy re quiring (e nde rgonic). S ta te me nts A a nd B a pply to both a na bolic a nd ca ta bolic proce s s e s , whe re a s C a nd E a pply only to ca ta bolic proce s s e s .

Corre ct a nswe r = E. Vigorously contra cting mus cle s hows a n incre a s e in the re duction of pyruva te to la cta te compa re d with re s ting s ke le ta l muscle . The le ve ls of a de nosine monophos pha te (AMP ) a nd re duce d nicotina mide a de nine dinucle otide (NADH) incre a s e , whe re a s cha nge in the conce ntra tion of fructos e 2,6-bis phos pha te is not a ke y re gula tory fa ctor in s ke le ta l mus cle . The ris e in the NADH to NAD+ ratio excee ds the oxida tive ca pa city of the re spiratory cha in.

Corre ct a ns we r = A. Glucose upta ke in the live r, bra in, muscle , a nd a dipose tis sue is down a concentra tion gradie nt, and the diffusion is fa cilitated by tis sue -spe cific glucos e tra nsporte rs (GLUTs ). In a dipos e a nd mus cle , ins ulin is re quire d for glucos e upta ke . Moving glucos e a ga ins t a concentra tion gradie nt re quires e nergy, a nd is s een with s odium-de pe nde nt glucos e tra ns porte r-1 (S GLT-1) of inte stina l mucos a l ce lls.

Corre ct a ns we r = He xokina s e . Km is tha t s ubs tra te conce ntra tion tha t give s 1 ⁄2 Vma x. Whe n blood glucos e conce ntra tion is 5 mM, he xokina s e (Km = 0.1 mM) will be s a tura te d, but glucokina s e (Km = 10 mM) will not.

Liga nde d G prote ins of the Gα i type inhibit a de nylyl cycla s e . If Gα i is inhibite d by toxin, a de nylyl cycla s e production of cyclic a de nos ine monophos pha te (cAMP ) is ina ppropria te ly a ctiva te d.

Tricarboxylic Acid Cycle and Pyruvate Dehydrogenas e Complex

9

I. OVERVIEW 6-P glucona te Ribulos e 5-P

The trica rboxylic a cid cycle ([TCA cycle ] a ls o ca lle d the citric a cid cycle or the Kre bs cycle ) pla ys s e ve ra l role s in me ta bolis m. It is the fina l pa thwa y whe re the oxida tive ca ta bolis m of ca rbohydra te s , a mino a cids , a nd fa tty a cids conve rge , the ir ca rbon s ke le tons be ing conve rte d to CO 2 (Figure 9.1). This oxida tion provide s e ne rgy for the production of the ma jority of a de nos ine triphos pha te (ATP ) in mos t a nima ls , including huma ns . The TCA cycle occurs tota lly in the mitochondria a nd is , the re fore , in clos e proximity to the re a ctions of e le ctron tra ns port (s e e p. 73), which oxidize the re duce d coe nzyme s (NADH a nd FADH2 ) produce d by the cycle . The TCA cycle is a n a e robic pa thwa y, be ca us e O 2 is re quire d a s the fina l e le ctron a cce ptor. Re a ctions s uch a s the ca ta bolis m of s ome a mino a cids ge ne ra te inte rme dia te s of the cycle a nd a re ca lle d a na ple rotic (“filling up”) re a ctions . The TCA cycle a ls o s upplie s inte rme dia te s for a numbe r of importa nt s ynthe tic re a ctions . For e xa mple , the cycle functions in the forma tion of glucos e from the ca rbon s ke le tons of s ome a mino a cids , a nd it provide s building blocks for the s ynthe s is of s ome a mino a cids (s e e p. 267) a nd he me (s e e p. 278). The re fore , this cycle s hould not be vie we d a s a clos e d circle but, ins te a d, a s a tra ffic circle with compounds e nte ring a nd le a ving a s re quire d.

Glycoge n UDP -Glucos e

6-P gluconola ctone

Ribos e 5-P

Ga la ctos e Ga la ctos e 1-P

Glucos e 1-P

Xylulos e 5-P

Glucos e 6-P

S e dohe ptulos e 7-P Erythros e 4-P

Fructos e 6-P

UDP -Ga la ctos e Glucos e Fructos e

Fructos e 1,6-bis -P Glyce ra lde hyde 3-P

Glyce ra lde hyde

Fructos e 1-P

Dihydroxya ce tone -P

Glyce ra lde hyde 3-P

Glyce rol-P

1,3-Bis phos phoglyce ra te

Glyce rol

3-P hos phoglyce ra te Tria cylglyce rol 2-P hos phoglyce ra te

Ala Cys Gly Ser Thr Try NH3

Fa tty a cid

Fa tty a cyl CoA P hos phoe nolpyruva te La cta te CO 2

CO 2

P yruva te CO 2

Ma lonyl CoA

Ace tyl CoA

Le u P he Tyr Trp Lys

Ace toa ce ta te

As n Ca rba moyl-P

β-Hydroxybutyra te As pa rta te

Citrulline

Oxalo ac e tate Malate

Ornithine

Argininos uccina te Fumarate Arginine

S uc c inate

Citrate Is oc itrate CO2 α-Ke to g lutarate CO2 S uc c inyl Co A

Gln P ro His Arg

Glu Me thylma lonyl CoA

Ure a Ile Me t Va l Thr

P he Tyr

P ropionyl CoA Ace tyl CoA Fa tty a cyl CoA (odd-numbe r ca rbons )

Ac e tyl Co A

II. REACTIONS OF THE CYCLE Oxalo ac e tate

In the TCA cycle , oxa loa ce ta te is firs t conde ns e d with a n a ce tyl group from a ce tyl coe nzyme A (CoA) a nd the n is re ge ne ra te d a s the cycle is comple te d (Figure 9.1). The re fore , the e ntry of one a ce tyl CoA into one round of the TCA cycle doe s not le a d to the ne t production or cons umption of inte rme dia te s . [Note : Two ca rbons e nte ring the cycle a s a ce tyl CoA a re ba la nce d by two CO 2 e xiting.]

Malate Fumarate S uc c inate

Citrate Is o c itrate CO2 α-Ke to g lutarate CO2 S uc c inyl Co A

A. Oxidative de c arbo xylatio n o f pyruvate

The ma jor s ource of a ce tyl CoA, the two-ca rbon s ubs tra te for the TCA cycle , is the oxida tive de ca rboxyla tion of pyruva te . P yruva te , the e nd product of a e robic glycolys is , mus t be tra ns porte d from the cytos ol into the mitochondrion. This is a ccomplis he d by a s pe cific tra ns porte r tha t fa cilita te s move me nt of pyruva te a cros s the inne r mitochondria l me mbra ne . Once in the mitochondria l ma trix, pyruva te

Fig ure 9.1 The trica rboxylic a cid cycle s hown a s a pa rt of the e s s e ntia l pa thwa ys of e ne rgy me ta bolis m. (S e e Figure 8.2, p. 92 for a more de ta ile d vie w of the me ta bolic ma p.) CoA = coe nzyme A. 109

110

9. Trica rboxylic Acid Cycle a nd P yruva te De hydroge na s e Comple x is conve rte d to a ce tyl CoA by the pyruva te de hydroge na s e comple x (P DH comple x), which is a multie nzyme comple x. [Note : S trictly s pe a king, the P DH comple x is not pa rt of the TCA cycle , but it s upplie s s ubs tra te for the cycle .] 1. Co mpo ne nt e nzyme s : The PDH complex is a protein aggregate of

multiple copies of three enzymes, pyruvate carboxylase (E1, sometimes ca lle d pyruvate dehydrogenase), dihydrolipoyl transace tylase (E2), and dihydrolipoyl dehydrogenase (E3). Each catalyzes a part of the ove rall rea ction (Figure 9.2). Their physical associa tion links the reactions in proper seque nce without the relea se of inte rmediate s. In addition to the enzymes participating in the conversion of pyruvate to acetyl CoA, the complex also contains two tightly bound regulatory e nzyme s, pyruvate dehydrogenase kinase (PDH kina se) and pyruvate dehydrogenase phosphata se (PDH phospha tase ). 2. Co e nzyme s : The P DH comple x conta ins five coe nzyme s tha t

a ct a s ca rrie rs or oxida nts for the inte rme dia te s of the re a ctions s hown in Figure 9.2. E1 re quire s thia mine pyrophos pha te (TPP ), E2 re quire s lipoic a cid a nd CoA, a nd E3 re quire s fla vin a de nine dinucle otide (FAD) a nd nicotina mide a de nine dinucle otide (NAD+). [Note : TP P, lipoic a cid, a nd FAD a re tightly bound to the e nzyme s a nd function a s coe nzyme s -pros the tic groups (s e e p. 54).] De ficie ncie s of thia mine or nia cin ca n ca us e s e rious ce ntra l ne rvous s ys te m proble ms . This is be ca us e bra in ce lls a re una ble to produce s ufficie nt ATP (via the TCA cycle ) if the P DH comple x is ina ctive . We rnicke -Kors a koff, a n e nce pha lopa thy-ps ychos is s yndrome due to thia mine de ficie ncy, ma y be s e e n with a lcohol a bus e .

O CH3 C COO-

O CH3 C ~ S L S H

TP P

P yruva te de ca rboxyla s e

1

2

CH3 CH TP P

Pyruvate is de c arbo xylate d to fo rm a hydro xye thyl de rivative bo und to the re ac tive c arbo n o f thiamine pyro pho s phate , the c o e nzyme o f p yru va te d e c a rb o xyla s e (E1). The hydro xye thyl inte rme diate is o xidize d by trans fe r to the dis ulfide fo rm o f lipo ic ac id c o vale ntly bo und to d ih yd ro lip o yl tra n s a c e tyla s e (E2).

O CH3 C ~ S CoA

Dihydrolipoyl tra ns a ce tyla s e

OH CO2

CoA oA

L

S

L

S

F FADH2

SH

FAD

Dihydrolipoyl Dihydrolip de hydroge nna s e

NAD+

NADH + H+

5

3

SH

4

The ac e tyl g ro up, bo und as a thio e s te r to the s ide c hain o f lipo ic ac id, is trans fe rre d to Co A.

The s ulfhydryl fo rm o f lipo ic ac id is o xidize d by FAD-de pe nde nt d ih yd ro lip o yl d e h yd ro g e n a s e (E3) le ading to the re g e ne ra ratio n o f o xidize d lipo ic ac id.

FADH2 o n E3 is re o xidize d to FAD as NAD+ is re duc e d to NADH + H+.

Fig ure 9.2 Me cha nis m of a ction of the pyruva te de hydroge na s e comple x. [Note : All the coe nzyme s of the comple x, e xce pt for lipoic a cid, a re de rive d from vita mins . TP P is from thia mine , FAD from ribofla vin, NAD from nia cin, a nd CoA from pa ntothe nic a cid.] TP P = thia mine pyrophos pha te ; L = lipoic a cid; CoA = coe nzyme A; FAD(H2 ) = fla vin a de nine dinucle otide ; NAD(H) = nicotina mide a de nine dinucle otide .

II. P yruva te De hydroge na s e Comple x

111

3. Re g ulatio n o f the pyruvate de hydro g e nas e c o mple x: Cova le nt

a ctivity of the α s ubunit of the dime ric E1 compone nt of the PDH comple x, a lthough ra re , is the mos t common bioche mica l ca us e of conge nita l la ctic a cidos is . This e nzyme de ficie ncy re s ults in a n ina bility to conve rt pyruva te to a ce tyl CoA, ca using pyruva te to be s hunte d to la cta te via la cta te de hydroge na s e (s e e p. 103). This cre a te s pa rticula r proble ms for the bra in, which relie s on the TCA cycle for mos t of its e ne rgy a nd is pa rticula rly s ens itive to a cidos is . S ymptoms a re va ria ble a nd include ne urode ge ne ra tion; mus cle s pa s ticity; a nd, in the ne ona ta l ons e t form, e a rly de a th. The ge ne for the α s ubunit is X linke d, a nd, be ca us e both ma le s a nd fe ma le s ma y be a ffe cte d, the de ficie ncy is cla s s ifie d a s X-linke d domina nt. Although the re is no prove n tre a tme nt for PDH comple x de ficie ncy, die ta ry re s triction of ca rbohydra te a nd s upple me nta tion with thia mine ma y re duce s ymptoms in s e le ct pa tie nts .

P yruva te de hydroge na s e comple x (ina ctive )

ADP

H2 O

Pyruvate ATP Ac e tyl Co A NADH

Ca 2+

P DH kina s e

+

4. Pyruvate de hydro g e nas e c o mple x de fic ie nc y: A de ficie ncy in the

P

+

modifica tions by the two re gula tory e nzyme s tha t a re pa rt of the comple x a lte rna te ly a ctiva te a nd ina ctiva te E1. The cyclic AMP – independent PDH kinase phosphorylates and, thereby, inactivates E1, whe re a s P DH phos pha ta s e de phos phoryla te s a nd a ctiva te s E1 (Figure 9.3). The kinase itself is allosterically activated by ATP, acetyl CoA, and NADH. Therefore, in the presence of these highenergy signals, the PDH complex is turned off. [Note: It is actually the rise in the ATP/ADP, NADH/NAD+, or acetyl CoA/CoA ratios that affects enzymic activity.] Pyruvate is a potent inhibitor of PDH kinase. Thus, if pyruvate concentrations are elevated, E1 will be maximally a ctive . Ca lcium (Ca 2+) is a s trong a ctiva tor of P DH phos pha ta s e , stimulating E1 activity. This is particularly important in skeletal muscle, whe re re lea se of Ca 2+ during contraction s timula tes the PDH complex and, thereby, energy production. [Note: Although covalent re gula tion by the kina s e a nd phos pha ta s e is prima ry, the complex is also subject to product (NADH and acetyl CoA) inhibition.]

P DH phos pha ta s e

P yruva te de hydroge na s e comple x (a ctive )

ATP

Pi

NADH Ac e tyl Co A

Pyruvate CO2

Fig ure 9.3 Re gula tion of pyruva te de hydroge na s e (P DH) comple x. [ de note s product inhibition.]

O CoA C CH3

+

Ac e tyl Co A

O O O C C O-O C CH 2

Oxalo ac e tate H2 O

Citra te s yntha s e

CoA CH2

O C O-

O HO C C O O -O C CH 2

Citrate Aconita s e

Le igh s yndrome (s uba cute ne crotizing e nce pha lomye lopa thy) is a ra re , progre s s ive , ne urode ge ne ra tive dis orde r ca us e d by de fe cts in mitochondria l ATP production, prima rily a s a re s ult of muta tions in ge ne s tha t code for prote ins of the P DH comple x, the e le ctron tra ns port cha in, or ATP s yntha s e . Both nucle a r a nd mitochondria l DNA ca n be a ffe cte d.

p. 101), pe nta va le nt a rs e nic (a rs e nate ) ca n inte rfe re with glycolys is a t the glyce ra lde hyde 3-phos pha te s te p, the re by de cre a s ing ATP production. “Ars e nic pois oning” is , howe ve r, due prima rily to inhibition of e nzyme s tha t re quire lipoic a cid a s a coe nzyme , including E2 of the PDH comple x, α -ke togluta ra te de hydroge na s e (s e e be low), a nd bra nche d-cha in α -ke to a cid de hydroge na s e (s e e p. 266). Ars e nite (the triva le nt form of a rs e nic) forms a sta ble comple x with the thiol (–S H) groups of lipoic a cid, ma king tha t compound una va ila ble to s e rve a s a coenzyme . Whe n it binds to lipoic a cid in the P DH comple x, pyruva te (a nd, cons e que ntly, la cta te )

O H C C OO -O C C H OH

Is o c itrate

+

NAD

ATP NADH ADP Ca 2+

+

5. Me c hanis m o f ars e nic po is o ning : As pre vious ly de s cribe d (s e e

CH2

O C O-

Is ocitra te de hydroge na s e

CH2

NADH + H+

O C O-

CO2

O CH2 -O C C O

α-Ke to g lutarate

Fig ure 9.4 Forma tion of α -ke togluta ra te from a ce tyl coe nzyme A (CoA) a nd oxa loa ce ta te . NAD(H) = nicotina mide a de nine dinucle otide

112

9. Trica rboxylic Acid Cycle a nd P yruva te De hydroge na s e Comple x a ccumula te s . As with P DH comple x de ficie ncy, this pa rticula rly a ffe cts the bra in, ca us ing ne urologic dis turba nce s a nd de a th.

O C O-

CH2

O CH2 -O C C O

B. S ynthe s is o f c itrate fro m ac e tyl c o e nzyme A and o xalo ac e tate

α-Ke to g lutarate CoA NAD+ NADH Suc c inyl Co A

CO2 NADH + H+

+

Ca 2+

α -Ke togluta ra te de hydroge na s e comple x

O O CH2 C O CoA C CH2

The conde ns a tion of a ce tyl CoA a nd oxa loa ce ta te (OAA) to form citra te (a trica rboxylic a cid) is ca ta lyze d by citra te s yntha s e (Figure 9.4). This a ldol conde ns a tion ha s a n e quilibrium fa r in the dire ction of citra te s ynthe s is . In huma ns , citra te s yntha s e is not a n a llos te ric e nzyme . It is inhibite d by its product, citra te . S ubs tra te a va ila bility is a nothe r me a ns of re gula tion for citra te s yntha s e . The binding of OAA ca us e s a conforma tiona l cha nge in the e nzyme tha t ge ne ra te s a binding s ite for a ce tyl CoA. [Note : Citra te , in a ddition to be ing a n inte rme dia te in the TCA cycle , provide s a s ource of a ce tyl CoA for the cytos olic s ynthe s is of fa tty a cids (s e e p. 183). Citra te a ls o inhibits phos phofructokina s e -1 (P FK-1), the ra te -limiting e nzyme of glycolys is (s e e p. 99), a nd a ctiva te s a ce tyl CoA ca rboxyla s e (the ra te -limiting e nzyme of fa tty a cid s ynthe s is ; s e e p. 183).]

S uc c inyl Co A GDP + P i S uccina te thiokina s e

GTP CoA O O CH2 C O -O C CH 2

C. Is o me rizatio n o f c itrate

Citra te is is ome rize d to is ocitra te by a conita s e (a conita te hydra ta s e ), a n Fe -S prote in (s e e Figure 9.4). [Note : Aconita s e is inhibite d by fluoroa ce ta te , a pla nt toxin tha t is us e d a s a pe s ticide . Fluoroa ce ta te is conve rte d to fluoroa ce tyl CoA, which conde ns e s with OAA to form fluorocitra te (a pote nt inhibitor of a conita s e ), re s ulting in citra te a ccumula tion.] D. Oxidative de c arbo xylatio n o f is o c itrate

S uc c inate FAD S uccina te de hydroge na s e

FADH2 O H

C O-

C O C -O C H

Is ocitra te de hydroge na s e ca ta lyze s the irre ve rs ible oxida tive de ca rboxyla tion of is ocitra te , yie lding the firs t of thre e NADH mole cule s produce d by the cycle a nd the firs t re le a s e of CO 2 (s e e Figure 9.4). This is one of the ra te -limiting s te ps of the TCA cycle . The e nzyme is a llos te rica lly a ctiva te d by ADP (a low-e ne rgy s igna l) a nd Ca 2+ a nd is inhibite d by ATP a nd NADH, le ve ls of which a re e le va te d whe n the ce ll ha s a bunda nt e ne rgy s tore s . E. Oxidative de c arbo xylatio n o f α-ke to g lutarate

Fumarate H2 O Fuma ra s e

H O HO C C O O -O C CH 2 L-Malate

Fig ure 9.5 Forma tion of ma la te from α -ke togluta ra te . NAD(H) = nicotina mide a de nine dinucle otide ; GDP = gua nos ine diphos pha te ; P = phos pha te ; CoA = coe nzyme A; FAD(H2 ) = fla vin a de nine dinucle otide .

The conve rs ion of α-ke togluta ra te to s uccinyl CoA is ca ta lyze d by the α-ke togluta ra te de hydroge na s e comple x, a prote in a ggre ga te of multiple copie s of thre e e nzyme s (Figure 9.5). The me cha nis m of this oxida tive de ca rboxyla tion is ve ry s imila r to tha t us e d for the conve rs ion of pyruva te to a ce tyl CoA by the PDH comple x. The re a ction re le a s e s the s e cond CO 2 a nd produce s the s e cond NADH of the cycle . The coe nzyme s re quire d a re TPP, lipoic a cid, FAD, NAD+, a nd CoA. Ea ch functions a s pa rt of the ca ta lytic me cha nis m in a wa y a na logous to tha t de s cribe d for the P DH comple x (s e e p. 110). The e quilibrium of the re a ction is fa r in the dire ction of s uccinyl CoA, a high-e ne rgy thioe s te r s imila r to a ce tyl CoA. α -Ke togluta ra te de hydroge na s e comple x is inhibite d by its products , NADH a nd s uccinyl CoA, a nd a ctiva te d by Ca 2+. Howe ve r, it is not re gula te d by phos phoryla tion/ de phos phoryla tion re a ctions a s de s cribe d for P DH comple x. [Note : α-Ke togluta ra te is a ls o produce d by the oxida tive de a mina tion (s e e p. 252) a nd tra ns amina tion of the a mino a cid gluta ma te (s e e p. 250).]

III. Ene rgy P roduce d by the TCA Cycle

113

F. Cle avag e o f s uc c inyl c o e nzyme A

S uccina te thiokina s e (a ls o ca lle d s uccinyl CoA s ynthe ta s e , na me d for the re ve rs e re a ction) cle a ve s the high-e ne rgy thioe s te r bond of s uccinyl CoA (s e e Figure 9.5). This re a ction is couple d to phos phoryla tion of gua nos ine diphos pha te (GDP ) to gua nos ine triphos pha te (GTP ). GTP a nd ATP a re e ne rge tica lly inte rconve rtible by the nucle os ide diphos pha te kina s e re a ction: GTP + ADP

→ ←

H O HO C C O O -O C CH 2 L-Malate

Ma la te de hydroge na s e

GDP + ATP

The ge ne ra tion of GTP by s uccina te thiokina s e is a nothe r e xa mple of s ubs tra te -le ve l phos phoryla tion (s e e p. 102). [Note : S uccinyl CoA is a ls o produce d from propionyl CoA de rive d from the me ta bolis m of fa tty a cids with a n odd numbe r of ca rbon a toms (s e e p. 193), a nd from the me ta bolis m of s e ve ra l a mino a cids (s e e pp. 265–266).] G. Oxidatio n o f s uc c inate

S uccina te is oxidize d to fuma ra te by s uccina te de hydroge na s e , a s FAD (its coe nzyme ) is re duce d to FADH 2 (s e e Figure 9.5). S uccina te de hydroge na s e is the only e nzyme of the TCA cycle tha t is e mbe dde d in the inne r mitochondria l me mbra ne . As s uch, it functions a s Comple x II of the e le ctron tra ns port cha in (s e e p. 75). [Note : FAD, ra the r tha n NAD+, is the e le ctron a cce ptor be ca us e the re ducing powe r of s uccina te is not s ufficie nt to re duce NAD+.]

+

NAD

O O C C O-

NADH + H+

O -O C CH 2 Oxalo ac e tate

Fig ure 9.6 Forma tion (re ge ne ra tion) of oxa loa ce ta te from ma la te . NAD(H) = nicotina mide a de nine dinucle otide .

H. Hydratio n o f fumarate Fuma ra te is hydra te d to ma la te in a fre e ly re ve rs ible re a ction ca ta lyze d by fuma ra s e (fuma ra te hydra ta s e ; s e e Figure 9.5). [Note : Fuma ra te is a ls o produce d by the ure a cycle (s e e p. 255), in purine s ynthe s is (s e e p. 294), a nd during ca ta bolis m of the a mino a cids phe nyla la nine a nd tyros ine (s e e p. 263).] I.

Oxidatio n o f malate

Ma la te is oxidize d to oxa loa ce ta te by ma la te de hydroge na s e (Figure 9.6). This re a ction produce s the third a nd fina l NADH of the cycle . The s ta nda rd fre e e ne rgy cha nge (∆G 0 ; s e e p. 70) of the re a ction is pos itive , but the re a ction is drive n in the dire ction of OAA by the highly e xe rgonic citra te s yntha s e re a ction. [Note : OAA is a ls o produce d by the tra ns a mina tion of the a mino a cid a s pa rtic a cid (se e p. 250).]

III. ENERGY PRODUCED BY THE CYCLE

Ene rgy-producing Numbe r of ATP re a ction produce d 3 NADH FADH 2

Two ca rbon a toms e nte r the cycle a s a ce tyl CoA a nd le a ve a s CO 2 . The cycle doe s not involve ne t cons umption or production of OAA or of a ny othe r inte rme dia te . Four pa irs of e le ctrons a re tra ns fe rre d during one turn of the cycle : thre e pa irs of e le ctrons re ducing thre e NAD+ to NADH a nd one pa ir re ducing FAD to FADH2 . Oxida tion of one NADH by the e le ctron tra ns port cha in le a ds to forma tion of a pproxima te ly thre e ATP , whe re a s oxida tion of FADH2 yie lds a pproxima te ly two ATP (s e e p. 77). The tota l yie ld of ATP from the oxida tion of one a ce tyl CoA is s hown in Figure 9.7. Figure 9.8 s umma rize s the re a ctions of the TCA cycle .

GDP + P i

3 NAD

+

FAD GTP

9 2 1 12 ATP /a ce tyl CoA oxidize d

Fig ure 9.7 Numbe r of ATP mole cule s produce d from the oxida tion of one mole cule of a ce tyl coe nzyme A (CoA) us ing both s ubs tra te -le ve l a nd oxida tive phos phoryla tion.

114

9. Trica rboxylic Acid Cycle a nd P yruva te De hydroge na s e Comple x

Two mo le c ule s o f CO2 are re le as e d.

A Two c arbo n ato ms e nte r the c yc le .

Ac Ac e tyl CoA

Citra te

Oxa loa c e ta te

Is oc itra te CO2 N AD +

N AD +

Ma la te

3 N ADH H α -Ke tog luta ra te

Fuma ra te

CO2 N AD +

F AD Suc c ina te

S c inyl CoA Suc CoA GTP (ATP)

F ADH 2

GDP + P i

Fo ur re duc e d S ubs trate -le ve l c o e nzyme mo le c ule s pho s pho rylatio n pro duc e d pe r acc e tyl o c c urs . Co A o xidize d to CO2 .

B

Ace tyl CoA Citra te s yntha s e

Citra te

Oxa loa ce ta te

Ma la te

Is ocitra te Is ocitra te de hydroge na s e

NADH ATP Fuma ra te NADH S uc c inyl Co A

+

ADP Ca 2+ α -Ke togluta ra te

+

α -Ke togluta ra te de hydroge na s e comple x

S uccina te S uccinyl CoA

Ca 2+

Fig ure 9.8 A. [Note: GTP a nd ATP are interconverted by nucleoside diphosphate kina se.] Production of re duced coenzymes, ATP, and CO 2 in the citric acid cycle. B. Inhibitors and activators of the cycle.

IV. REGULATION OF THE CYCLE In contra s t to glycolys is , which is re gula te d prima rily by P FK-1, the TCA cycle is controlle d by the re gula tion of s e ve ra l e nzyme s (s e e Figure 9.8). The mos t importa nt of the s e re gula te d e nzyme s a re thos e tha t ca ta lyze re a ctions with highly ne ga tive ∆G 0 : citra te s yntha s e , is ocitra te de hydroge na s e , a nd α -ke togluta ra te de hydroge na s e comple x. Re ducing e quiva le nts ne e de d for oxida tive phos phoryla tion a re ge ne ra te d by the P DH comple x a nd the TCA cycle , a nd both proce s s e s a re upre gula te d in re s pons e to a de cre a s e in the ra tio of ATP to ADP .

V. CHAPTER S UMMARY Pyruvate is o xidative ly de c arbo xylate d by p yru va te d e h yd ro g e n a s e (P DH) c o m p le x , producing ac e tyl c o e nzyme A (Co A), which is the

ma jor fue l for the trica rboxylic a cid cycle ([TCA cycle ] Figure 9.9). This multie nzyme comple x re quire s five coe nzyme s : thiamine pyro pho s phate , lipo ic ac id , flavin ade nine dinuc le o tide (FAD), nic o tinamide ade nine dinuc le o tide (NAD+), a nd Co A. P DH comple x is re gula te d by cova le nt modifica tion of E1 (pyruva te de ca rboxyla s e ) by P DH kina s e a nd P DH phos pha ta s e : phos phoryla tion inhibits E1. P DH kina s e is a llos te rica lly a ctiva te d by ATP , a ce tyl CoA, a nd NADH a nd inhibite d by pyruva te . The phos pha ta s e is a ctiva te d by Ca 2+ . P DH c o m p le x de fic ie nc y is the mos t common bioche mica l ca us e of c o ng e nital lac tic ac ido s is . The ce ntra l ne rvous s ys te m is pa rticula rly a ffe cte d in this X-linke d do minant dis orde r. Ars e nic po is o ning ca us e s ina ctiva tion of the P DH comple x by binding to lipoic a cid. Citrate is s ynthe s ize d from o xalo ac e tate a nd ac e tyl Co A by c itra te s yn th a s e . This e nzyme is s ubje ct to product inhibition by citra te . Citra te is is ome rize d to is o c itrate by a c o n ita s e (a c o n ita te h yd ra ta s e ). Is o c itrate is oxida tive ly de ca rboxyla te d by is o c itra te d e h yd ro g e n a s e to α-ke to g lutarate , producing CO2 a nd NADH. The e nzyme is inhibite d by ATP a nd NADH, a nd a ctiva te d by ADP a nd Ca 2+ . α-Ke to g lutarate is oxida tive ly de ca rboxyla te d to s uc c inyl Co A by the α-ke to g lu ta ra te d e h yd ro g e n a s e c o m p le x , producing CO2 a nd NADH. The e nzyme is ve ry s imila r to the P DH comple x a nd us e s the s a me coe nzyme s . α -Ke togluta ra te de hydroge na s e comple x is a ctiva te d by Ca +2 a nd inhibite d by NADH a nd s uccinyl CoA but is not cova le ntly re gula te d. S uc c inyl Co A is cle a ve d by s u c c in a te th io kin a s e (a ls o ca lle d s u c c in yl Co A s yn th e ta s e ), producing s uc c inate a nd GTP . This is a n e xa mple of s ubs trate -le ve l pho s pho rylatio n . S uc c inate is oxidize d to fumarate by s u c c in a te d e h yd ro g e n a s e , producing FADH2 . Fumarate is hydra te d to malate by fu m a ra s e (fu m a ra te h yd ra ta s e ), a nd malate is oxidize d to o xalo ac e tate by m a la te d e h yd ro g e n a s e , producing NADH. Thre e NADH, o ne FADH2 , a nd o ne GTP (whos e te rmina l phos pha te ca n be tra ns fe rre d to ADP by nucle os ide diphos pha te kina s e , producing ATP ) a re produce d by one round of the TCA cycle . The ge ne ra tion of a ce tyl CoA by the oxida tion of pyruva te via the P DH comple x a ls o produce s a n NADH. Oxida tion of the NADH a nd FADH2 by the e le ctron tra ns port cha in yie lds 14 ATP . An a dditiona l ATP (GTP ) come s from s ubs tra te le ve l phos phoryla tion in the TCA cycle . The re fore , a tota l of 15 ATP a re produce d from the comple te mitochondria l oxida tion of pyruva te to CO 2 .

V. Cha pte r S umma ry

115

Func tio n o f the TCA c yc le

Re g ulatio n o f the TCA c yc le cons is ts of

Carbo hydrate Amino ac ids Fatty ac ids

Dire c t re g ulatio n o f e nzyme ac tivitie s by pro duc t inhibitio n o r by allo s te ric e ffe c to rs s uc h as ATP, ADP, and NADH

Ac e tyl Co A Citra te

Oxa loa ce ta te

provide s for

re s ponds to e ithe r

Is ocitra te Ma la te

Fuma ra te

Oxidatio n o f ac e tyl Co A

CO2 α -Ke togluta ra te

S uccina te

Hig h-e ne rg y s tate

2CO2

Ace tyl CoA

Is ocitra te NAD+

ATP

a nd

a nd

ADP o r P i

ADP o r P i

le a ds to

le a ds to

Oxidatio n o f NADH le a ds to

NAD+ Succinate Succinyl CoA FAD GTP

GDP + Pi

N ADH

C O 2 produce s

NADH/NAD+ le a ds to

NADH and FADH2 le a d to

ADP AT P

ATP

Oxidative pho s pho rylatio n le a ds to

Oxidatio n o f NADH

α -Ke togluta ra te

Fumara te

F ADH 2

cha ra cte rize d by

le a ds to

CO2 NAD+

1 /2 O 2

cha ra cte rize d by

Oxidative pho s pho rylatio n

Citra te

Oxa loa ce ta te Ma la te

Lo w-e ne rg y s tate

to

CO2

S uccinyl CoA

Indire c t re g ulatio n thro ug h o blig ato ry c o upling o f o xidatio n with pho s pho rylatio n

TCA c yc le ac tivity

produce s

le a ds to

NADH/NAD+ le a ds to

TCA c yc le ac tivity

C o n c e p t c o n n e c t

O x id a t iv e p h o s p h o r y la t io n H2O

Fo rmatio n o f ATP

N AD +

F ADH

a s a re s ult of

a s a re s ult of

Oxidative pho s pho rylatio n

S ubs trate le ve l pho s pho rylatio n

Oxa loa ce ta te

provide s for

S ynthe tic re ac tio ns

Citra te Is ocitra te

for e xa mple

Ma la te

Amino ac ids

Fuma ra te

S uccina te

6

Co n c e p t c o n n e c t

Ace tyl CoA

Amino ac ids

Bioenergetics and Oxidative Phos phorylation

Amino ac ids

P yruva te

Gluc o s e

GTP

CO2

Amino ac ids

α -Ke togluta ra te

Gluconeogens is

10

Co nve rs io n o f amino ac ids to g luc o s e

CO2 S uccinyl CoA

Amino ac ids

Co nc e pt c o nne c t

Fig ure 9.9 Ke y conce pt ma p for the trica rboxylic a cid (TCA) cycle . CoA = coe nzyme A; NAD(H) = nicotina mide a de nine dinucle otide ; FAD(H2 ) = fla vin a de nine dinucle otide ; GDP = gua nos ine diphos pha te ; GTP = gua nos ine triphos pha te ; ADP = a de nos ine diphos pha te ; P i = inorga nic phos pha te .

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9. Trica rboxylic Acid Cycle a nd P yruva te De hydroge na s e Comple x

S tudy Que s tio ns Choos e the ONE be s t a ns we r. 9.1 The conve rs ion of pyruva te to a ce tyl coe nzyme A a nd CO 2 : A. involve s the pa rticipa tion of lipoic a cid. B. is a ctiva te d whe n pyruva te de ca rboxyla s e of the pyruva te de hydroge na s e (P DH) comple x is phos phoryla te d by PDH kina s e in the pre se nce of ATP . C. is re ve rs ible . D. occurs in the cytos ol. E. re quire s the coe nzyme biotin. 9.2 Which one of the following conditions de cre a s e s the oxida tion of a ce tyl coe nzyme A by the citric a cid cycle? A. B. C. D.

A high a va ila bility of ca lcium A high a ce tyl CoA/CoA ra tio A low ATP /ADP ra tio A low NAD+/NADH ra tio

9.3 The following is the s um of thre e s te ps in the citric a cid cycle .

Corre ct a ns we r = A. Lipoic a cid is a n inte rme dia te a cce ptor of the a ce tyl group forme d in the re a ction. P yruva te de hydroge na s e comple x ca ta lyze s a n irre ve rs ible re a ction tha t is inhibite d whe n the de ca rboxyla s e compone nt is phos phoryla te d. The e nzyme comple x is loca te d in the mitochondria l ma trix. Biotin is utilize d by ca rboxyla s e s , not de ca rboxyla s e s .

Corre ct a ns we r = D. A low NAD+/NADH ra tio limits the ra te s of the NAD+-re quiring de hydroge na s e s. High a va ila bility of ca lcium a nd s ubs tra te (a ce tyl CoA), a nd a low ATP/ADP ra tio stimula te s the cycle .

Corre ct a ns we r = B. S uccina te + NAD+ + FAD + H2 O → oxa loa ce ta te + NADH + FADH2

A + B + FAD + H2 O → C + FADH2 + NADH Choos e the le tte re d a ns we r tha t corre s ponds to the mis s ing “A,” “B,” a nd “C” in the e qua tion. Re a cta nt A A. S uccinyl CoA B. S uccina te C. Fuma ra te D. S uccina te E. Fuma ra te

Re a cta nt B GDP NAD+ NAD+ NAD+ GTP

Re a cta nt C S uccina te Oxa loa ce ta te Oxa loa ce ta te Ma la te Ma la te

9.4 A 1-month-old ma le s hows ne urologic proble ms a nd la ctic a cidos is . Enzyme a s s a y for pyruva te de hydroge na s e (P DH) comple x a ctivity on e xtra cts of culture d s kin fibrobla s ts s howe d 5% of norma l a ctivity with a low conce ntra tion of thia mine pyrophos pha te (TP P ), but 80% of norma l a ctivity whe n the a s s a y conta ine d a thous a nd-fold highe r conce ntra tion of TP P . Which one of the following s ta te me nts conce rning this pa tie nt is corre ct? A. Adminis tra tion of thia mine is e xpe cte d to re duce his s e rum la cta te le ve l a nd improve his clinica l s ymptoms . B. A high ca rbohydra te die t would be e xpe cte d to be be ne ficia l for this pa tie nt. C. C itra te p ro d u c tio n fro m a e ro b ic g lyc o lys is is e xpe cte d to be incre a s e d. D. P DH kina s e , a re gula tory e nzyme of the P DH comple x, is e xpe cte d to be a ctive . 9.5 Which coe nzyme -cos ubs tra te is us e d by the de hydroge na s e s of both glycolys is a nd the trica rboxylic a cid cycle ?

Corre ct a ns we r = A. The pa tie nt a ppe a rs to ha ve a thia mine -re s pons ive pyruva te de hydroge na s e (P DH) comple x de ficie ncy. The pyruva te de ca rboxyla s e (E1) compone nt of the P DH comple x fa ils to bind thia mine pyrophos pha te a t low conce ntra tion, but s hows s ignifica nt a ctivity a t a high conce ntra tion of the coe nzyme . This muta tion, which a ffe cts the Km of the e nzyme for the coe nzyme , is pre s e nt in s ome , but not a ll, ca s e s of P DH comple x de ficie ncy. Be ca us e the P DH comple x is a n inte gra l pa rt of ca rbohydra te me ta bolis m, a die t low in ca rbohydra te s would be e xpe cte d to blunt the e ffe cts of the e nzyme de ficie ncy. Ae robic glycolys is ge ne ra te s pyruva te , the s ubs tra te of the P DH comple x. De cre a s e d a ctivity of the comple x de cre a s e s production of a ce tyl coe nzyme A, a s ubs tra te for citra te s yntha s e . P DH kina s e is a llos te rica lly inhibite d by pyruva te a nd, the re fore , is ina ctive .

O xid iz e d n ic o tin a mid e a de n in e d in u c le o tid e (NAD+) is us e d by glyce ra lde hyde 3-phos pha te de hydroge na s e of glycolys is a nd by is ocitra te d e h ydro g e n a s e , α -ke to g lu ta ra te d e h yd rog e na s e , a nd ma la te de hydroge na s e of the trica rboxylic a cid cycle .

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Gluc o ne o g e ne s is I. OVERVIEW 6-P glucona te

Some tis s ue s , s uch a s the bra in, re d blood ce lls (RBCs ), kidne y me dulla , le ns a nd corne a of the e ye , te s te s , a nd e xe rcis ing mus cle , re quire a con tinuous s upply of glucos e a s a me ta bolic fue l. Liver glycoge n, a n e s s e n tia l pos tpra ndia l s ource of glucos e , ca n me e t the s e ne e ds for only 10–18 hours in the a bs e nce of die ta ry inta ke of ca rbohydra te (s e e p. 329). During a prolonge d fa s t, howe ve r, he pa tic glycoge n s tore s a re de ple te d, a nd glucos e is forme d from nonca rbohydra te pre curs ors s uch a s la cta te , pyruva te , glyce rol (de rive d from the ba ckbone of tria cylglyce rols ; s e e p. 190), a nd α ke to a cids (de rive d from the ca ta bolis m of glucoge nic a mino a cids ; s e e p. 261). The forma tion of glucos e doe s not occur by a s imple re ve rs a l of glycolys is , be ca us e the ove ra ll e quilibrium of glycolys is s trongly fa vors pyruva te forma tion. Ins te a d, glucos e is s ynthe s ize d by a s pe cia l pa thwa y, glucone oge ne s is , which re quire s both mitochondria l a nd cytos olic e nzyme s . During a n ove rnight fa s t, a pproxima te ly 90% of glucone oge ne s is occurs in the live r, with the re ma ining 10% occurring in the kidne ys . Howe ve r, during prolonge d fa s ting, the kidne ys be come ma jor glucos e producing orga ns , contributing a n e s tima te d 40% of the tota l glucos e production. Figure 10.1 s hows the re lations hip of glucone o ge ne s is to othe r e s s e ntia l pa thwa ys of e ne rgy me ta bolis m.

Ribulos e 5-P

Glycoge n UDP -Glucos e

6-P gluconola ctone

Ribos e 5-P

Ga la ctos e Ga la ctos e 1-P

Glucos e 1-P

Xylulos e 5-P

Glucos e 6-P

S e dohe ptulos e 7-P Erythros e 4-P

Fructos e 6-P

UDP -Ga la ctos e Glucos e Fructos e

Fructos e 1,6-bis -P Glyce ra lde hyde 3-P

Glyce ra lde hyde 3-P

Glyce ra lde hyde

Fructos e 1-P

Dihydroxya ce tone -P Glyce rol-P

1,3-bis -P hos phoglyce ra te

Glyce rol

3-P hos phoglyce ra te Tria cylglyce rol 2-P hos phoglyce ra te

Ala Cys Gly Ser Thr Try NH3

Fa tty a cid

Fa tty a cyl CoA P hos phoe nolpyruva te La cta te CO 2

CO 2

P yruva te CO 2

Ma lonyl CoA

Ace tyl CoA

Ace toa ce ta te

Le u P he Tyr Trp Lys

As n Ca rba moyl-P

β-Hydroxybutyra te As pa rta te

Citrulline

Ornithine

Oxa loa ce ta te

Citra te

Ma la te

Is ocitra te CO 2 α -Ke togluta ra te

Argininos uccina te Fuma ra te

CO 2 S uccinyl CoA

S uccina te

Arginine

Gln P ro His Arg

Glu Me thylma lonyl CoA

Ure a Ile Me t Va l Thr

P he Tyr

Glucos e 6-P

4

P ropionyl CoA Ace tyl CoA Fa tty a cyl CoA (odd-numbe r ca rbons )

Glucos e

Fructos e 6-P

3 Fructos e 1,6-bis phos pha te

II. S UBS TRATES FOR GLUCONEOGENES IS

Glyce ra lde hyde 3-P

Dihydroxya ce tone -P

1,3-Bis phos phoglyce ra te

Glucone oge nic pre curs ors a re mole cule s tha t ca n be us e d to produce a ne t s ynthe s is of glucos e . The mos t importa nt glucone oge nic pre s curors a re glyce rol, la cta te , a nd the α ke to a cids obta ine d from the me ta bolis m of glucoge nic a mino a cids . [Note : Ala nine , which dire ctly give s ris e to pyruva te , is a n importa nt e xa mple of a glucoge nic a mino a cid.] A. Glyc e ro l Glyce rol is re le a s e d during the hydrolys is of tria cylglyce rols in a di pos e tis s ue (s e e p. 190) a nd is de live re d by the blood to the live r. Glyce rol is phos phoryla te d by glyce rol kina s e to glyce rol phos pha te , which is oxidize d by glyce rol phos pha te de hydroge na s e to dihydroxya ce tone phos pha te , a n inte rme dia te of glycolys is . [Note : Adipocyte s ca nnot phos phoryla te glyce rol be ca us e the y e s s e ntia lly la ck glyce rol kina s e .]

3-P hos phoglyce ra te 2-P hos phoglyce ra te P hos phoe nolpyruva te

2 La cta te CO 2

P yruva te

1

Oxa loa ce ta te

Fig ure 10.1 The gluconeogenesis pathway shown a s one of the e s s e ntia l pa thwa ys of e ne rgy me ta bolis m. The numbe re d reactions are unique to gluconeogenesis. (See Figure 8.2, p. 92, for a more detailed map of metabolism.) P = phosphate.

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10. Glucone oge ne s is B. Lac tate LIVER

Gluc o s e

Lac tate

BLOOD Lac tate

Gluc o s e

Lac tate MUS CLE Gluc o s e

Fig ure 10.2 The inte rtis s ue Cori cycle . [Note : Diffus ion of la cta te a cros s me mbra ne s is fa cilita te d by a tra ns port prote in.]

La cta te is re le a s e d into the blood by e xe rcis ing s ke le ta l mus cle a nd by ce lls tha t la ck mitochondria s uch a s RBCs . In the Cori cycle , bloodborne glucos e is conve rte d by e xe rcis ing mus cle to la cta te , which diffus e s into the blood. This la cta te is ta ke n up by the live r a nd re conve rte d to glucos e , which is re le a s e d ba ck into the circula tion (Figure 10.2). C. Amino ac ids Amino a cids de rive d from hydrolys is of tis s ue prote ins a re the ma jor s ource s of glucos e during a fa s t. The me ta bolis m of the glu coge nic a mino a cids ge ne ra te s α ke to a cids . α Ke to a cids , s uch a s α ke togluta ra te ca n e nte r the trica rboxylic a cid (TCA) cycle a nd form oxa loa ce ta te (OAA), a dire ct pre curs or of phos phoe nolpyruva te (P EP ). [Note : Ace tyl coe nzyme A (CoA) a nd compounds tha t give ris e only to a ce tyl CoA (for e xa mple , a ce toa ce ta te a nd a mino a cids s uch a s lys ine a nd le ucine ) ca nnot give ris e to a ne t s ynthe s is of glu cos e . This is due to the irre ve rs ible na ture of the pyruva te de hydroge na s e (P DH) re a ction, which conve rts pyruva te to a ce tyl CoA (s e e p. 109). The s e compounds give ris e ins te a d to ke tone bodie s (s e e p. 195) a nd a re , the re fore , te rme d ke toge nic.]

III. REACTIONS UNIQUE TO GLUCONEOGENES IS S e ve n glycolytic re a ctions a re re ve rs ible a nd a re us e d in the s ynthe s is of glucos e from la cta te or pyruva te . Howe ve r, thre e of the re a ctions a re irre ve rs ible a nd mus t be circumve nte d by four a lte rna te re a ctions tha t e ne rge tica lly fa vor the s ynthe s is of glucos e . The s e re a ctions , unique to glucone oge ne s is , a re de s cribe d be low. A. Carbo xylatio n o f pyruvate The firs t “roa dblock” to ove rcome in the s ynthe s is of glucos e from pyruva te is the irre ve rs ible conve rs ion in glycolys is of P EP to pyru va te by pyruva te kina s e (P K). In glucone oge ne s is , pyruva te is firs t ca rboxyla te d by pyruva te ca rboxyla s e to OAA, which is the n con ve rte d to P EP by the a ction of P EP -ca rboxykina s e (Figure 10.3). 1. Bio tin, a c o e nzyme : P yruva te ca rboxyla s e re quire s biotin (s e e

p. 381) cova le ntly bound to the ε a mino group of a lys ine re s idue in the e nzyme (s e e Figure 10.3). Hydrolys is of ATP drive s the forma tion of a n e nzyme –biotin–CO 2 inte rme dia te , which s ubs e que ntly ca rboxyla te s pyruva te to form OAA. [Note : HCO 3 – is the s ource of the CO 2 .] The pyruva te ca rboxyla s e re a ction occurs in the mitochondria of live r a nd kidne y ce lls a nd ha s two purpos e s : to provide a n importa nt s ubs tra te for glucone oge ne s is a nd to provide OAA tha t ca n re ple nis h the TCA cycle inte rme dia te s tha t ma y be come de ple te d, de pe nding on the s ynthe tic ne e ds of the ce ll. Mus cle ce lls a ls o conta in pyruva te ca rboxyla s e but us e the OAA produce d only for the re ple nis hme nt (a na ple rotic) purpos e a nd do not s ynthe s ize glucos e . Pyruvate carboxylase is one of several carboxylases that require biotin. Others include acetyl CoA carboxylase (p. 183), propionyl CoA carboxylase (p. 193), and methylcrotonyl CoA carboxylase (p. 266).

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III. Re a ctions Unique to Glucone oge ne s is

P yru va te c a rb o xyla s e (with c o vale ntly attac he d bio tin)

119



1

CO2 fro m HCO3 is ac tivate d and trans fe rre d by p yru va te c a rb o xyla s e to its bio tin pro s the tic g ro up.

2

The e nzyme the n trans fe rs the CO2 to pyruvate , g e ne rating o xalo ac e tate .

+ ATP

Lys ine re s idue o f e nzyme

Ac e tyl Co A



HCO3

O -O C

HN

NH

Bio tin S

O -O C

Pyruvate

NH

O

O O C C O-

CH3

NH O

O O C C O-

ADP + P i

O

O N

NH

3

S

Oxalo ac e tate c anno t c ro s s the mito c ho ndrial me mbrane s o it is re duc e d to malate that c an.

NH

CH2

O

Oxalo ac e tate (OAA) NADH + H+

O

HN

NH

MDm NAD+

S

Malate

MITO CHO NDR ION CYTO S OL

O P

O C C O-

CO2

GDP

NADH + H+

GTP

4

NAD+

CH2 Pho s pho e no lpyruvate (PEP)

OAA

MDc

Malate

In the c yto s o l, malate is re o xidize d to o xalo ac e tate , whic h is o xidative ly de c arbo xylate d to pho s pho e no lpyruvate by P EP c a rb o xykin a s e .

Fig ure 10.3 Ca rboxyla tion of pyruva te to OAA, followe d by re duction of OAA to ma la te for tra ns fe r to the cys tol a nd s ubs e que nt de ca rboxyla tion to P EP . [Note : OAA ca n a ls o be conve rte d to P EP or a s pa rta te for tra ns fe r to the cytos ol.] MDm = mitochondria l ma la te de hydroge na s e ; MDc = cytos olic ma la te de hydroge na s e . 2. Allo s te ric re g ulatio n: P yruva te ca rboxyla s e is a llos te rica lly a cti

va te d by a ce tyl CoA. Ele va te d le ve ls of a ce tyl CoA in mitochon dria s igna l a me ta bolic s ta te in which the incre a s e d s ynthe s is of OAA is re quire d. For e xa mple , this occurs during fa s ting, whe n OAA is us e d for the s ynthe s is of glucos e by glucone oge ne s is in the live r a nd kidne y. Conve rs e ly, a t low le ve ls of a ce tyl CoA, pyruva te ca rboxyla s e is la rge ly ina ctive , a nd pyruva te is prima rily oxidize d by the P DH comple x to produce a ce tyl CoA tha t ca n be furthe r oxidize d by the TCA cycle (s e e p. 109). B. Trans po rt o f o xalo ac e tate to the c yto s o l OAA must be converted to PEP for gluconeogenesis to continue. The enzyme that ca talyzes this reaction is found in both the mitochondria and the cytosol in humans. The PEP generated in the mitochondria is transported to the cytosol by a specific tra nsporte r, whereas that gener ate d in the cytosol requires the transport of OAA from the mitochondria to the cytosol. However, OAA is unable to be transported across the inne r mitochondrial membrane, so it must first be reduced to malate by mitochondrial mala te dehydrogenase (MD). Malate can be transported from the mitochondria to the cytosol, whe re it is reoxidized to OAA by cytosolic MD as nicotinamide ade nine dinucleotide (NAD+) is re duced (s e e Figure 10.3). The NADH produce d is us e d in the re duction of 1,3 bisphosphoglycera te to glycera ldehyde 3 phosphate (see p. 101), a ste p common to both glycolys is and glucone ogene sis. [Note : OAA a ls o ca n be conve rte d to a s pa rta te , which is tra ns porte d out of the mitochondria.]

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10. Glucone oge ne s is C. De c arbo xylatio n o f c yto s o lic o xalo ac e tate

AMP Fruc to s e 2,6-bis pho s phate

OAA is de ca rboxyla te d a nd phos phoryla te d to P EP in the cytos ol by P EP -ca rboxykina s e (a ls o re fe rre d to a s P EP CK). The re a c tion is drive n by hydrolys is of gua nos ine triphos pha te ([GTP ] s e e Figure 10.3). The combine d a ctions of pyruva te ca rboxyla s e a nd P EP -ca rboxykina s e provide a n e ne rge tica lly fa vora ble pa thwa y from pyruva te to P EP . P EP is the n a cte d on by the re a ctions of glycolys is running in the re ve rs e dire ction until it be come s fructos e 1,6 bis phos pha te .

H

H H C O

P

H C OH

C O HO C H H C OH

P

H2 O

H C OH

H C OH H C O

C O HO C H H C OH

P

P

Fructos e 1,6- H C O bis phos pha ta s e H

H

Fruc to s e 1,6bis pho s phate

The pa iring of ca rboxyla tion with de ca rboxyl a tion, a s s e e n in glucone oge ne s is , drive s re a c tio n s th a t wo u ld o th e rwis e b e e n e rg e tic a lly unfa vora ble . A s imila r s tra te gy is us e d in fa tty a cid s ynthe s is (s e e pp. 183–184).

Fruc to s e 6pho s phate

Fig ure 10.4 De phos phoryla tion of fructos e 1,6bis phos pha te . AMP = a de nos ine monophos pha te ; P = phos pha te .

D. De pho s pho rylatio n o f fruc to s e 1,6-bis pho s phate Hydrolys is of fructos e 1,6 bis phos pha te by fructos e 1,6-bis phos pha ta s e , found in live r a nd kidne y, bypa s s e s the irre ve rs ible phos phofructokina s e -1 (P FK-1) re a ction, a nd provide s a n e ne rge ti ca lly fa vora ble pa thwa y for the forma tion of fructos e 6 phos pha te (Figure 10.4). This re a ction is a n importa nt re gula tory s ite of glucone oge ne s is . 1. Re gulatio n by e ne rgy le ve ls within the c e ll: Fructos e 1,6-bis phos

pha ta s e is inhibite d by e le va te d le ve ls of a de nos ine monophos Gluc ag o n (hig h)

Ins ulin (lo w)

Re ce ptor

CELL MEMBRANE

Ad e n ylyl c yc la s e ATP c AMP

CYTOS OL

Re ce ptor

1

Hig h g luc ag o n/ins ulin ratio c aus e s e le vate d c AMP and inc re as e d le ve ls o f ac tive p ro te in kin a s e A.

2

Ac tive p ro te in kin a s e A F r u c t o s e 6 -p h o s p h a t e

G lu c o n e o g e n e s is Glucos e 6-P

Glucos e

Fruc to s e 6-pho s phate

P

P

P Fr u c t o s e 6 -P

ADP

FBP -1

ATP

F r u c t o s e 1 ,6 -b is -P

Glyce ra lde hyde 3-P

DHAP

Inc re as e d p ro te in kin a s e A ac tivity favo rs the pho s pho rylate d fo rm o f the bifunc tio nal P FK-2/FBP -2.

PFK-2 (inactive)

H2O

P FK-2 (a ctive )

FBP -2 (a ctive )

FBP -2 (ina ctive )

1,3-Bis phos phoglyce ra te 3-P hos phoglyce ra te

F B P -1

P Fruc to s e

Bifunctiona l e nzyme

2,6-bis pho s phate

2-P hos phoglyce ra te

De c re as e d le ve ls o f fruc to s e 2,6-bis pho s phate detec re as e the inhibitio n o f FBP -1, whic h P yruva le ads to an inc re as e d rate o f g luc o ne o g e ne s is .

P hos phoe nolpyruva te

4

La cta te

3

Pho s pho rylatio n o f the P FK-2 do main inac tivate s it, allo wing the FBP -2 do main to be ac tive .

Fig ure 10.5 Effe ct of e le va te d gluca gon on the intra ce llula r conce ntra tion of fructos e 2,6-bis phos pha te in the live r. cAMP = cyclic AMP ; P FK-2 = phos phofructokina s e -2; FBP -2 = fructos e 2,6-bis phos pha ta s e ; FBP -1 = fructos e 1,6-bis phos pha ta s e ; P = phos pha te .

tahir99-VRG & vip.persianss.ir

IV. Re gula tion of Glucone oge ne s is pha te (AMP ), which s igna l a n “e ne rgy poor” s ta te in the ce ll. Conve rs e ly, high le ve ls of ATP a nd low conce ntra tions of AMP s timula te glucone oge ne s is , a n e ne rgy re quiring pa thwa y. 2. Re g ulatio n by fruc to s e 2,6-bis pho s phate : Fructos e 1,6-bis phos -

pha ta s e is inhibite d by fructos e 2,6 bis phos pha te , a n a llos te ric e ffe ctor whos e conce ntra tion is influe nce d by the ins ulin to glu ca gon ra tio: whe n gluca gon is high, the e ffe ctor is not ma de a nd, thus , the phos pha ta s e is a ctive . (Figure 10.5). [Note : The s igna ls tha t inhibit (low e ne rgy, high fructos e 2,6 bisphos pha te ) or a ctiva te (high e ne rgy, low fructos e 2,6 bis phos pha te ) glucone oge ne s is ha ve the oppos ite e ffe ct on glycolys is , providing re ciproca l control of the pa thwa ys tha t s ynthe s ize a nd oxidize glucos e (s e e p. 100).] E. De pho s pho rylatio n o f g luc o s e 6-pho s phate Hydrolys is of glucos e 6 phos pha te by glucos e 6-phos pha ta s e bypa s s e s the irre ve rs ible he xokina s e /glucokina s e re a ction a nd provide s a n e ne rge tica lly fa vora ble pa thwa y for the forma tion of fre e glucos e (Figure 10.6). Live r a nd kidne y a re the only orga ns tha t re le a s e fre e glucos e from glucos e 6 phos pha te . This proce s s a ctua lly re quire s a comple x of two prote ins : glucos e 6 phos pha te transloca se, which transports glucose 6 phosphate across the endo pla s mic re ticula r (ER) me mbra ne , a nd the e nzyme glucos e 6-phos pha ta s e (found only in glucone oge nic ce lls ), which re move s the phos pha te , producing fre e glucos e (s e e Figure 10.6). [Note : The s e ER me mbra ne prote ins a re a ls o re quire d for the fina l s te p of gly coge n de gra da tion (s e e p. 130). Type Ia a nd lb glycoge n s tora ge dis e a s e , ca us e d by de ficie ncie s in the phos pha ta s e a nd the tra ns fe ra s e , re s pe ctive ly, a re cha ra cte rize d by s e ve re fa s ting hypogly ce mia , be ca us e fre e glucos e is una ble to be produce d from e ithe r glucone oge ne s is or glycoge nolys is .] S pe cific glucos e tra ns porte rs (GLUTs) a re re sponsible for moving free glucose into the cytosol and then into blood. [Note: Glucose 6 phosphate translocase moves inor ganic phosphate out of the ER a s it moves glucose 6 phosphate in.] F. S ummary o f the re ac tio ns o f g lyc o lys is and g luc o ne o g e ne s is Of the 11 re a ctions re quire d to conve rt pyruva te to fre e glucos e , 7 a re ca ta lyze d by re ve rs ible glycolytic e nzyme s (Figure 10.7). The irre ve rs ible re a ctions of glycolys is ca ta lyze d by he xokina s e /glucokina s e, P FK-1, a nd P K a re circumve nte d by glucos e 6-phos pha ta s e, fructos e 1,6-bis phos pha ta s e , a nd pyruva te ca rboxyla s e /P EP -ca rboxykina s e . In glucone oge ne s is , the e quilibria of the 7 re ve rs ible re a ctions of glycolys is a re pus he d in fa vor of glucos e s ynthe s is a s a re s ult of the e s s e ntia lly irre ve rs ible forma tion of P EP, fructos e 6 phos pha te , a nd glucos e ca ta lyze d by the glucone oge nic e nzyme s . [Note : The s toichi ome try of glucone oge ne s is from pyruva te couple s the cle a va ge of s ix high e ne rgy phos pha te bonds a nd the oxida tion of two NADH with the forma tion of e a ch mole cule of glucos e (s e e Figure 10.7).]

IV. REGULATION OF GLUCONEOGENES IS The mome nt to mome nt re gula tion of glucone oge ne s is is de te rmine d prima rily by the circula ting le ve l of gluca gon a nd by the a va ila bility of glucone oge nic s ubs tra te s . In a ddition, s low a da ptive cha nge s in

121

O

O

C H

C H

H C OH HO C H H C OH

H C OH HO C H H C OH

P

H2 O

H C OH

H C OH P

H C O H

H C OH

Glucos e 6phos pha ta s e

H D-Gluc o s e

Gluc o s e 6pho s phate

Fig ure 10.6 De phos phoryla tion of glucos e 6phos pha te a llows re le a s e of fre e glucos e from the live r a nd kidne y into blood. P = phos pha te .

4

Glucos e 6-P

Glucos e

Fructos e 6-P

3 Fructos e 1,6-bis -P Glyce ra lde hyde 3-P

Dihydroxya ce tone -P

2 NAD+ 2 NADH + 2H+

Pi

2 1,3-Bis phos phoglyce ra te 2 ADP 2 ATP 2 3-P hos phoglyce ra te 2 GDP

2 2-P hos phoglyce ra te 2 P hos phoe nolpyruva te 2 P yruva te

2 CO 2

1

2 ADP + 2 P i 2 ATP

2 Oxa loa ce ta te 2 GTP

Fig ure 10.7 S umma ry of the re a ctions of glycolys is a nd glucone oge ne s is , s howing the e ne rgy re quire me nts of glucone oge ne s is . The numbe re d re a ctions a re unique to glucone oge ne s is . P = phos pha te ; GDP = gua nos ine diphos pha te ; GTP = gua nos ine triphos pha te ; NAD(H) = nicotina mide a de nine dinucle otide .

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122

10. Glucone oge ne s is e nzyme a ctivity re s ult from a n a lte ra tion in the ra te of e nzyme s ynthe s is or de gra da tion or both. [Note : Hormona l control of the glucore gula tory s ys te m is pre s e nte d in Cha pte r 23.]

Gluc ag o n

Re ce ptor

A. Gluc ag o n

Ade nylyl cycla s e

ATP

c AMP + PP i

Gluc o s e

1. Chang e s in allo s te ric e ffe c to rs : Gluca gon lowe rs the le ve l of

Ac tive p ro te in kin a s e A PEP ADP P ATP

ADP

P yruva te kina s e (a ctive )

P yruva te kina s e (ina ctive )

ATP

This pe ptide hormone from the α ce lls of pa ncre a tic is le ts (s e e p. 313) s timula te s glucone oge ne s is by thre e me cha nis ms .

OAA

Pyruvate

Fig ure 10.8 Cova le nt modifica tion of pyruva te kina s e re s ults in ina ctiva tion of the e nzyme . [Note : Only the he pa tic is ozyme is s ubje ct to cova le nt re gula tion.] OAA = oxa loa ce ta te ; P EP = phos phoe nolpyruva te ; cAMP = cyclic AMP ; P P i = pyrophos pha te ; P = phos pha te .

fructos e 2,6 bis phos pha te , re s ulting in a ctiva tion of fructos e 1,6-bis phos pha ta s e a nd inhibition of P FK-1, thus fa voring gluco ne oge ne s is ove r glycolys is (s e e Figure 10.5). [Note : S e e p. 99 for the role of fructos e 2,6 bis phos pha te in the re gula tion of glycolys is .] 2. Co vale nt mo dific atio n o f e nzyme ac tivity: Gluca gon binds its

G prote in–couple d re ce ptor (s e e p. 95) a nd, via a n e le va tion in cyclic AMP (cAMP ) le ve l a nd cAMP -de pe nde nt prote in kina s e a ctivity, s timula te s the conve rs ion of he pa tic P K to its ina ctive (phos phoryla te d) form. This de cre a s e s the conve rs ion of P EP to pyruva te , which ha s the e ffe ct of dive rting P EP to the s ynthe s is of glucos e (Figure 10.8). 3. Induc tio n o f e nzyme s ynthe s is : Gluca gon incre a s e s the tra n

s cription of the ge ne for P EP -ca rboxykina s e , the re by incre a s ing the a va ila bility of this e nzyme a s le ve ls of its s ubs tra te ris e during fa s ting. [Note : Glucocorticoids a ls o incre a s e e xpre s s ion of the ge ne , whe re a s ins ulin de cre a s e s e xpre s s ion.] B. S ubs trate availability The a va ila bility of glucone oge nic pre curs ors , pa rticula rly glucoge nic a mino a cids , s ignifica ntly influe nce s the ra te of glucos e s ynthe s is . De cre a s e d le ve ls of ins ulin fa vor mobiliza tion of a mino a cids from mus cle prote in a nd provide the ca rbon s ke le tons for glucone oge n e s is . The ATP a nd NADH coe nzyme s cos ubs tra te s re quire d for glu cone oge ne s is a re prima rily provide d by the ca ta bolis m of fa tty a cids .

Pyruvate

P DH c o m p le x



+

P yru va te c a rb o xyla s e

Oxalo ac e tate

Ac e tyl Co A

TCA c yc le

C. Allo s te ric ac tivatio n by ac e tyl c o e nzyme A

Fatty ac ids

Gluc o s e

Allos te ric a ctiva tion of he pa tic pyruva te ca rboxyla s e by a ce tyl CoA occurs during fa s ting. As a re s ult of incre a s e d lipolys is in a dipos e tis s ue , the live r is floode d with fa tty a cids (s e e p. 330). The ra te of for ma tion of a ce tyl CoA by β oxida tion of the s e fa tty a cids e xce e ds the ca pa city of the live r to oxidize it to CO 2 a nd H2 O. As a re s ult, a ce tyl CoA a ccumula te s a nd a ctiva te s pyruva te ca rboxyla s e. [Note : Ace tyl CoA inhibits the P DH comple x (by a ctiva ting PDH kina s e ; s e e p. 111). Thus , this s ingle compound ca n dive rt pyruva te toward glucone oge n e s is a nd a wa y from the TCA cycle (Figure 10.9).] D. Allo s te ric inhibitio n by ade no s ine mo no pho s phate

Fig ure 10.9 Acetyl coenzyme A (CoA) diverts pyruvate away from oxidation and toward gluconeogenesis. PDH = pyruvate dehydrogenase; TCA = tricarboxylic acid.

Fructos e 1,6-bis phos pha ta s e is inhibite d by AMP —a compound tha t a ctiva te s P FK-1. This re s ults in a re ciproca l re gula tion of glycolys is a nd glucone oge ne s is s e e n pre vious ly with fructos e 2,6 bis phos pha te (s e e p. 121). [Note : Ele va te d AMP , thus , s timula te s pa thwa ys tha t oxidize nutrie nts to provide e ne rgy for the ce ll.]

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V. CHAPTER S UMMARY Gluc o ne o g e nic pre c urs o rs include the inte rme diate s o f g lyc o lys is a nd the tric arbo xylic ac id c yc le , g lyc e ro l re le a s e d during the hydrolys is of tria cylglyce rols in a dipos e tis s ue , lac tate re le a s e d by ce lls tha t la ck mitochondria a nd by e xe rcis ing s ke le ta l mus cle , a nd α-ke to ac ids de rive d from the me ta bolis m of glucoge nic a mino a cids (Figure

10.10). Se ve n of the re a ctions of glycolys is a re re ve rs ible a nd a re us e d for glucone oge ne s is in the live r a nd kidne ys . Thre e re a ctions a re phys iolog ic ally irre ve rs ible a nd mus t be circumve nte d. The s e re a ctions a re ca ta lyze d by the glycolytic e nzyme s p yru va te kin a s e , p h o s p h o fru c to kin a s e , a nd h e xo kin a s e . Pyruvate is conve rte d to oxa loa c e ta te a nd the n to phos pho e nolpyruvate (PEP ) by p yruva te c a rboxyla s e a nd PEP -c a rb oxykina s e . The ca rboxyla s e re quire s bio tin a nd ATP a nd is a llos te rica lly a ctiva te d by ac e tyl c o e nzyme A. P EP -ca rboxykina s e re quire s GTP . The tra ns cription of its ge ne is incre a s e d by glucagon a nd the glucocorticoids a nd de cre a s e d by ins ulin. Fruc tos e 1,6-bis pho s phate is conve rte d to fruc to s e 6-pho s phate by fru c to s e 1,6-b is p h o s p h a ta s e . This e nzyme is inhibite d by e le va te d le ve ls of AMP a nd ac tivate d whe n ATP le ve ls a re e le va te d. The e nzyme is a ls o inhibite d by fruc to s e 2,6-bis pho s phate , the prima ry a llos te ric a ctiva tor of glycolys is . Gluc o s e 6-pho s phate is conve rte d to g luc o s e by g luc o s e 6-phos pha ta s e . This e nzyme of the e ndopla s mic re ticula r me mbra ne is re quire d for the fina l s te p in gluco ne oge ne s is a s we ll a s hepa tic a nd re na l glycoge n de gra da tion. Its de ficie ncy re s ults in s e ve re , fa s ting hypoglyce mia .

Re g ulatio n o f g luc o ne o g e ne s is during fas ting

S ubs trate s fo r g luc o ne o g e ne s is

Fas ting s tate

Gluc o ne o g e ne s is cons is ts of La c t a t e RED BLOOD CELL MUS CLE (e xe rcis ing)

Re le as e o f fatty ac ids fro m adipo s e tis s ue

Pyruvate 1 Oxalo ac e tate

Re g ulate d s te ps Fatty ac id o xidatio n in the live r

provide

PEP

1,3-BPG

Carbo n s ke le to ns fo r de no vo s ynthe s is o f g luc o s e

G 3-P

cons is t of

3-PG

1 P yru va te c a rb o xyla s e 2

ADIP OS E

F 1,6-bis -P 2

Blo o d g luc o s e Re le as e o f g luc ag o n

Glyc e ro l, lac tate whic h e nte r dire c tly into g luc o ne o g e ne s is

F 6-P G 6-P

c AMP

a nd

Gluc o s e

Ac e tyl Co A in live r

Fru c to s e 1,6-b is p h o s p h a ta s e

+

G ly c e r o l

+

2-PG

P ro te in kin a s e A ac tivity

Amino ac ids whos e me ta bolis m conve rge s on the

Ace tyl CoA

Oxa loa ceta te

Citra te

TCA c yc le

Tricarboxylic Acid Cycle and Pyruvate Dehydrogenas e Complex

Is ocitra te Ma la te

Am in o a c id s

Fuma ra te

S uccina te

CO2 α -Ke togluta ra te

Am in o a c id s

CO2 S uccinyl CoA

Am in o a c id s

Fruc to s e 2,6-bis pho s phate

Co nc e pt c o nne c t

9

P yruvate kinas e ac tivity Co nve rs io n o f pho s pho e no lpyruvate (PEP) to pyruvate PEP is dive rte d to the s ynthe s is o f g luc o s e

Fig ure 10.10 Ke y conce pt ma p for glucone oge ne s is . TCA = trica rboxylic a cid. CoA = coe nzyme A; cAMP = cyclic a de nos ine monophos pha te ; P = phos pha te ; P G = phos phoglyce ra te ; BP G = bis phos phoglyce ra te .

124

10. Glucone oge ne s is

S tudy Que s tio ns Choos e the ONE be s t a ns we r. 10.1 Which one of the following s ta te me nts conce rning glucone oge ne s is is corre ct? A. It is a n e ne rgy producing (e xe rgonic) proce s s . B. It is importa nt in ma inta ining blood glucos e during a fa s t. C. It is inhibite d by a fa ll in the ins ulin to gluca gon ra tio. D. It occurs in the cytos ol of mus cle ce lls . E. It us e s ca rbon s ke le tons provide d by fa tty a cid de gra da tion. 10.2 Which re a ction in the dia gra m be low would be inhib ite d in the pre s e nce of la rge a mounts of a vidin, a n e gg white prote in tha t binds a nd s e que s te rs biotin?

Corre ct a ns we r = C. P yruva te is ca rboxyla te d to oxa loa ce ta te by pyruva te ca rboxyla s e , a biotin re quiring e nzyme . B (P DH comple x) re quire s thia mine pyrophos pha te , lipoic a cid, FAD, coe n zyme A, NAD; D (tra ns a mina s e ) re quire s pyri doxa l phos pha te ; E (la cta te de hydroge na s e ) re quire s NADH.

P hos phoe nolpyruva te A La cta te

E

P yruva te D

Ala nine

B

Ace tyl coe nzyme A

C Oxa loa ce ta te

10.3 Which one of the following re a ctions is unique to glu cone oge ne s is ? A. B. C. D.

1,3 Bis phos phoglyce ra te → 3 phos phoglyce ra te La cta te → pyruva te Oxa loa ce ta te → phos phoe nolpyruva te P hos phoe nolpyruva te → pyruva te

10.4 Us e the cha rt be low to s how the e ffe ct of a de nos ine monophos pha te (AMP ) a nd fructos e 2,6 bis phos pha te on the lis te d e nzyme s of glucone oge ne s is a nd glycolys is .

Enzyme

Correct answer = B. During a fast, glycogen stores a re de ple te d, a nd glucone oge ne s is ma inta ins blood glucos e . Glucone oge ne s is is a n e ne rgy re quiring (e nde rgonic) pa thwa y (both ATP a nd GTP get hydrolyzed) that occurs in liver, with kid ne y be coming a ma jor glucos e producing orga n in prolonge d fa s ting. It utilize s both mitochon dria l a nd cytosolic e nzymes. Gluconeoge ne sis is stimula te d by a fa ll in the ins ulin/gluca gon ra tio. Fatty acid degradation yields acetyl coenzyme A (CoA), which cannot be converted to glucose. This is be ca use the re is no ne t ga in of ca rbons from acetyl CoA in the tricarboxylic acid cycle, and the pyruva te de hydroge na s e re a ction is phys iologi cally irreversible. It is the carbon skeletons of most amino acids that are gluconeogenic.

Fructos e 2,6 bis phos pha te

AMP

Fructos e 1,6 bis phos pha ta s e P hos phofructokina s e 1 10.5 The me ta bolis m of e tha nol by a lcohol de hydroge na s e produce s re duce d nicotina mide a de nine dinu cle otide (NADH). Wha t e ffe ct is the cha nge in the NAD+/NADH ra tio e xpe cte d to ha ve on glucone oge n e s is ? Expla in. 10.6 Give n tha t a ce tyl coe nzyme A ca nnot be a s ubs tra te for glucone oge ne s is , why is its production in fa tty a cid oxida tion e s s e ntia l for glucone oge ne s is ?

Corre ct a ns we r = C. The othe r re a ctions a re common to both glucone oge ne s is a nd glycolys is .

Both fructos e 2,6 bis phos pha te a nd a de nos ine monophos pha te downre gula te glucone oge ne s is through inhibition of fructos e 1,6 bis phos pha ta s e a nd upre gula te glycolys is through a ctiva tion of phos phofructokina s e 1. This re s ults in re ciproca l re gula tion of the two pa thwa ys .

The incre a s e in NADH a s e tha nol is oxidize d will de cre a s e the a va ila bility of oxa loa ce ta te (OAA) be ca us e the re ve rs ible oxida tion of ma la te to OAA by ma la te de hydroge na s e of the trica rbox ylic a cid cycle is drive n in the re ve rs e dire ction by the high a va ila bility of NADH. Additiona lly, the re ve rs ible re du c tion of pyruva te to la c ta te by la cta te de hydroge na s e of glycolys is is drive n in the forwa rd dire ction by NADH. Thus , two importa nt glucone oge nic s ubs tra te s , OAA a nd pyruva te , a re de cre a s e d a s a re s ult of the incre a s e in NADH during e tha nol me ta bolis m. This re s ults in a de cre a s e in glucone oge ne s is .

Ace tyl coe nzyme A inhibits the pyruva te de hy droge na s e comple x a nd a ctiva te s pyruva te ca r boxyla s e , pus hing pyruva te to glucone oge ne s is a nd a wa y from oxida tion.

Glyc o g e n Me tabo lis m

11

I. OVERVIEW 6-P Glucona te

A cons ta nt s ource of blood glucos e is a n a bs olute re quire me nt for huma n life . Glucos e is the gre a tly pre fe rre d e ne rgy s ource for the bra in, a nd the re quire d e ne rgy s ource for ce lls with fe w or no mitochondria s uch a s ma ture re d blood ce lls . Glucos e is a ls o e s s e ntia l a s a n e ne rgy s ource for e xe rcis ing mus cle , whe re it is the s ubs tra te for a na e robic glycolys is . Blood glucos e ca n be obta ine d from thre e prima ry s ource s : the die t, de gra da tion of glycoge n, a nd glucone oge ne s is . Die ta ry inta ke of glucos e a nd glucos e pre curs ors , s uch a s s ta rch (a polys a ccha ride ), dis a ccha ride s , a nd monos a ccha ride s , is s pora dic a nd, de pe nding on the die t, is not a lwa ys a re lia ble s ource of blood glucos e . In contra s t, glucone oge ne s is (s e e p. 117) ca n provide s us ta ine d s ynthe s is of glucos e , but it is s ome wha t s low in re s ponding to a fa lling blood glucos e le ve l. The re fore , the body ha s de ve lope d me cha nis ms for s toring a s upply of glucos e in a ra pidly mobiliza ble form, na me ly, glycoge n. In the a bs e nce of a die ta ry s ource of glucos e , this s uga r is ra pidly re le a s e d from live r a nd kidne y glycoge n. S imila rly, mus cle glycoge n is e xte ns ive ly de gra de d in e xe rcis ing mus cle to provide tha t tis s ue with a n importa nt e ne rgy s ource . Whe n glycoge n s tore s a re de ple te d, s pe cific tis s ue s s ynthe s ize glucos e de novo, us ing a mino a cids from the body’s prote ins a s a prima ry s ource of ca rbons for the glucone oge nic pa thwa y. Figure 11.1 s hows the re a ctions of glycoge n s ynthe s is a nd de gra da tion a s pa rt of the e s s e ntia l pa thwa ys of e ne rgy me ta bolis m.

II. S TRUCTURE AND FUNCTION OF GLYCOGEN The ma in s tore s of glycoge n a re found in s ke le ta l mus cle a nd live r, a lthough mos t othe r ce lls s tore s ma ll a mounts of glycoge n for the ir own us e . The function of mus cle glycoge n is to s e rve a s a fue l re s e rve for the s ynthe s is of a de nos ine triphos pha te (ATP ) during mus cle contra ction. Tha t of live r glycoge n is to ma inta in the blood glucos e conce ntra tion, pa rticula rly during the e a rly s ta ge s of a fa s t (Figure 11.2; a ls o s e e p. 329). [Note : Live r glycoge n ca n ma inta in blood glucos e for 10–18 hours .] A. Amo unts o f live r and mus c le g lyc o g e n Approxima te ly 400 g of glycoge n ma ke up 1%–2% of the fre s h we ight of re s ting mus cle , a nd a pproxima te ly 100 g of glycoge n ma ke up to 10% of the fre s h we ight of a we ll-fe d a dult live r. Wha t limits the production of glycoge n a t the s e le ve ls is not cle a r. Howe ve r, in s ome

Ribulos e 5-P

Glycoge n UDP -Glucos e

6-P Gluconola ctone

Ribos e 5-P

Ga la ctos e Ga la ctos e 1-P

Glucos e 1-P

Xylulos e 5-P

Glucos e 6-P

S e dohe ptulos e 7-P Erythros e 4-P

Fructos e 6-P

UDP -Ga la ctos e Glucos e Fructos e

Fructos e 1,6-bis -P Glyce ra lde hyde 3-P

Glyce ra lde hyde 3-P

Glyce ra lde hyde

Fructos e 1-P

Dihydroxya ce tone -P Glyce rol-P

1,3-bis -P hos phoglyce ra te

Glyce rol

3-P hos phoglyce ra te Tria cylglyce rol 2-P hos phoglyce ra te

Ala Cys Gly Ser Thr Try NH3

Fa tty a cyl CoA

Fa tty a cid

P hos phoe nolpyruva te La cta te CO 2

CO 2

P yruva te CO 2

Ma lonyl CoA

Ace tyl-CoA

Le u P he Tyr Trp Lys

Ace toa ce ta te

As n Ca rba moyl-P

β-Hydroxybutyra te As pa rta te

Citrulline

Ornithine

Oxa loa ce ta te

Citra te

Ma la te

Is ocitra te CO 2 α -Ke togluta ra te

Argininos uccina te Fuma ra te Arginine

S uccina te

CO 2 S uccincyl CoA

Gln P ro His Arg

Glu Me thylma lonyl CoA

Ure a Ile Me t Va l Thr

P he Tyr

P ropionyl CoA Ace tyl CoA Fa tty a cyl CoA (odd-numbe r ca rbons )

Glyc o g e n UDP-Gluc o s e Gluc o s e 1-P Gluc o s e 6-P

Gluc o s e

Fig ure 11.1 Glycoge n s ynthe s is a nd de gra da tion s hown a s a pa rt of the e s s e ntia l pa thwa ys of e ne rgy me ta bolis m (s e e Figure 8.2, p. 92, for a more de ta ile d vie w of the ove ra ll re a ctions of me ta bolis m). P = phos pha te ; UDP = uridine diphos pha te .

125

126

11. Glycoge n Me ta bolis m

MUS CLE

Glyc o g e n Glyc o g e n

glycoge n s tora ge dis e a s e s ([GSDs ] s e e Figure 11.8), the a mount of glycoge n in the live r a nd/or mus cle ca n be s ignifica ntly highe r. [Note : In the body, mus cle ma s s is gre a te r tha n live r ma s s . Cons e que ntly, mos t of the body’s glycoge n is found in mus cle .]

Gluc o s e 6-P

B. S truc ture o f g lyc o g e n Pi Gluc o s e

Gluc o s e 6-P

LIVER

BLOOD GLUCOS E

ENERGY

Glycoge n is a bra nche d-cha in polys a ccha ride ma de e xclus ive ly from α -D-glucos e . The prima ry glycos idic bond is a n α (1→4) linka ge . Afte r a n a ve ra ge of e ight to te n glucos yl re s idue s , the re is a bra nch conta ining a n α (1→6) linka ge (Figure 11.3). A s ingle glycoge n mole cule ca n ha ve a mole cula r we ight of up to 10 8 Da . The s e polyme rs of glucos e e xis t in dis cre te cytopla s mic gra nule s tha t a ls o conta in mos t of the e nzyme s ne ce s s a ry for glycoge n s ynthe s is a nd de gra da tion. C. Fluc tuatio n o f g lyc o g e n s to re s

Fig ure 11.2 Functions of mus cle a nd live r glycoge n. P = phos pha te ; P i = inorga nic phos pha te .

Live r glyc og e n s tore s in c re a s e during th e we ll-fe d s ta te (s e e p. 323) a nd a re de ple te d during a fa s t (s e e p. 329). Mus cle glycoge n is not a ffe cte d by s hort pe riods of fa s ting (a fe w da ys ) a nd is only mode ra te ly de cre a s e d in prolonge d fa s ting (we e ks ). Mus cle glycoge n is s ynthe s ize d to re ple nis h mus cle s tore s a fte r the y ha ve be e n de ple te d following s tre nuous e xe rcis e . [Note : Glycoge n s ynthe s is a nd de gra da tion go on continuous ly. The diffe re nce s be twe e n the ra te s of the s e two proce s s e s de te rmine the le ve ls of s tore d glycoge n during s pe cific phys iologic s ta te s .]

III. S YNTHES IS OF GLYCOGEN (GLYCOGENES IS ) Glycoge n is s ynthe s ize d from mole cule s of α -D-glucos e . The proce s s occurs in the cytosol and require s energy supplied by ATP (for the phos phoryla tion of glucos e ) a nd uridine triphos pha te (UTP ). A. S ynthe s is o f uridine dipho s phate g luc o s e

C O H2

O

H

O

O

H

α (1 →6) g lyc o s idic bo nd C

H

O H2

O

O

H

O

O

H H

CH2 OH O O

OH

CH2

CH2 OH O

O O

OH

O

O

α (1 →4) g lyc o s idic bo nds

OH

O OH

OH

α -D-Glucos e a tta che d to uridine diphos pha te (UDP ) is the s ource of a ll the glucos yl re s idue s tha t a re a dde d to the growing glycoge n mole cule . UDP -glucos e (Figure 11.4) is s ynthe s ize d from glucos e 1-phos pha te a nd UTP by UDP -glucos e pyrophos phoryla s e (Figure 11.5). P yrophos pha te (P P i), the s e cond product of the re a ction, is hydrolyze d to two inorga nic phos pha te s (P i) by pyrophos pha ta s e . The hydrolys is is e xe rgonic, e ns uring tha t the UDP -glucos e pyrophos phoryla s e re a ction proce e ds in the dire ction of UDP -glucos e production. [Note : Glucos e 1-phos pha te is ge ne ra te d from glucos e 6-phos pha te by phos phoglucomuta s e . Glucos e 1,6-bis phos pha te is a n obliga tory inte rme dia te in this re ve rs ible re a ction (Figure 11.6).] B. S ynthe s is o f a prime r to initiate g lyc o g e n s ynthe s is

OH

Fig ure 11.3 Bra nche d s tructure of glycoge n, s howing α (1 → 4) a nd α (1 → 6) glycos idic bonds .

Glycoge n s yntha s e ma ke s the α (1→4) linka ge s in glycoge n. This e nzyme ca nnot initia te cha in s ynthe s is us ing fre e glucos e a s a n a cce ptor of a mole cule of glucos e from UDP -glucos e . Ins te a d, it ca n only e longa te a lre a dy e xis ting cha ins of glucose a nd, the re fore , re quire s a prime r. A fra gme nt of glycoge n ca n s e rve a s a prime r in ce lls whos e glycoge n s tore s a re not tota lly de ple te d. In the a bs e nce

III. S ynthe s is of Glycoge n (Glycoge ne s is )

127

of a glycoge n fra gme nt, a prote in ca lle d glycoge nin ca n s e rve a s a n a cce ptor of glucos e re s idue s from UDP -glucos e (s e e Figure 11.5). The s ide -cha in hydroxyl group of a s pe cific tyros ine in the prote in s e rve s a s the s ite a t which the initia l glucos yl unit is a tta che d. Be ca us e the re a ction is ca ta lyze d by glycoge nin its e lf via a utoglucos yla tion, glycoge nin is a n e nzyme . Glycoge nin the n ca ta lyze s the tra ns fe r of the ne xt fe w mole cule s of glucos e from UDP -glucos e , producing a s hort, α (1→4)-linke d glucos yl cha in. This s hort cha in s e rve s a s a prime r tha t is a ble to be e longa te d by glycoge n s yntha s e a s de s cribe d be low [Note : Glycoge nin s ta ys a s s ocia te d with a nd forms the core of a glycoge n gra nule .]

UDP-Gluc o s e O C

H H CH2 OH N C O C H H C C H N O O H O C C OH H O P O P O CH2 O HO C C OOH O H C C H H H H C C OH OH

Gluc o s e

Uridine dipho s phate (UDP)

C. Elo ng atio n o f g lyc o g e n c hains by g lyc o g e n s ynthas e Fig ure 11.4 The s tructure of UDP -glucos e , a nucle otide s uga r.

Elonga tion of a glycoge n cha in involve s the tra ns fe r of glucos e from UDP -glucos e to the nonre ducing e nd of the growing cha in, forming a ne w glycos idic bond be twe e n the a nome ric hydroxyl group of ca rbon 1 of the a ctiva te d glucos e a nd ca rbon 4 of the a cce pting glucos yl re s idue (s e e Figure 11.5). [Note : The nonre ducing e nd of a ca rbohydra te cha in is one in which the a nome ric ca rbon of the te rmina l s uga r is linke d by a glycos idic bond to a nothe r compound, ma king the te rmina l s uga r nonre ducing (s e e p. 84).] The e nzyme re s pons ible for ma king the α (1→4) linka ge s in glycoge n is glycoge n s yntha s e . [Note : The UDP re le a s e d whe n the ne w α (1→4) glycos idic bond is ma de ca n be phos phoryla te d to UTP by nucle os ide diphos pha te kina s e (UDP + ATP → ← UTP + ADP ; s e e p. 296).] Gluc o s e 6-pho s phate Tyro s ine

P hos phoglucomuta s e

Gluc o s e 1-pho s phate UTP UDP -glucos e pyrophos phoryla s e

HO

UDP-g luc o s e (UDP - )

Glyc o g e n in

PP i UDP

P yrophos pha ta s e

H2 O

O UDP-g luc o s e (UDP - )

2 Pi

α(1→6) Glycoge n s yntha s e

o n

UDP

m

α(1→4) bo nds o

n

m

l

k

j

i

h

g

f

e

d

c

b

bo nd

Bra nching e nzyme

a

O

4:6 tra ns fe ra s e

l k j

i

h

f

e

d

c

b

a

O

Furthe r e lo ng atio n at the no nre duc ing e nds by g lyc o g e n s yn th a s e , making α(1→4) bo nds .

NONREDUCING ENDS

Furthe r branc hing , making α(1→6) bo nds .

GLYCOGEN

Fig ure 11.5 Glycoge n s ynthe s is . UTP = uridine triphos pha te ; UDP = uridine diphos pha te ; P P i = pyrophos pha te ; P i = inorga nic phos pha te .

128

11. Glycoge n Me ta bolis m D. Fo rmatio n o f branc he s in g lyc o g e n

Phos phog lu c o m u ta s e

If no othe r s ynthe tic e nzyme a cte d on the cha in, the re s ulting s tructure would be a line a r (unbra nche d) cha in of glucos yl re s idue s a tta che d by α (1→4) linka ge s . S uch a compound is found in pla nt tis s ue s a nd is ca lle d a mylos e . In contra s t, glycoge n ha s bra nche s loca te d, on a ve ra ge , e ight glucos yl re s idue s a pa rt, re s ulting in a highly bra nche d, tre e -like s tructure (s e e Figure 11.3) tha t is fa r more s oluble tha n the unbra nche d a mylos e . Bra nching a ls o incre a s e s the numbe r of nonre ducing e nds to which ne w glucos yl re s idue s ca n be a dde d (a nd a ls o, a s de s cribe d la te r, from which the s e re s idue s ca n be re move d), the re by gre a tly a cce le ra ting the ra te a t which glycoge n s ynthe s is ca n occur a nd dra ma tica lly incre a s ing the s ize of the glycoge n mole cule .

Gluc o s e 6- P

P Phos phog lu c o m u ta s e

Gluc o s e 1,6- P

Phos phog lu c o m u ta s e

Gluc o s e 1- P

P

P

Fig ure 11.6 Inte rconve rs ion of glucos e 6phos pha te a nd glucos e 1-phos pha te by phos phoglucomuta s e . P = phos pha te .

HO

HO

O OH

HO

HO

O OH

O OH

HO

O OH

O

O OH

OH

OH OH

Glyc o g e n c hain

P LP Glycoge n phos phoryla s e

HO

The de gra da tive pa thwa y tha t mobilize s s tore d glycoge n in live r a nd s ke le ta l mus cle is not a re ve rs a l of the s ynthe tic re a ctions . Ins te a d, a s e pa ra te s e t of cytos olic e nzyme s is re quire d. Whe n glycoge n is de gra de d, the prima ry product is glucos e 1-phos pha te , obta ine d by bre a king α (1→4) glycos idic bonds . In a ddition, fre e glucos e is re le a s e d from e a ch α (1→6)–linke d glucos yl re s idue (bra nch point).

O 2–

OH

e nds ha s be e n a ccomplis he d, the ir te rmina l s ix to e ight glucos yl re s idue s ca n be re move d a nd us e d to ma ke a dditiona l bra nche s .

IV. DEGRADATION OF GLYCOGEN (GLYCOGENOLYSIS)

Pi

HO

bra nching e nzyme , a mylo-α (1→4)→α (1→6)-tra ns glucos ida s e . This e nzyme re move s a s e t of s ix to e ight glucos yl re s idue s from the nonre ducing e nd of the glycoge n cha in, bre a king a n α (1→4) bond to a nothe r re s idue on the cha in, a nd a tta che s it to a nonte rmina l glucos yl re s idue by a n α (1→6) linka ge , thus functioning a s a 4:6 tra ns fe ra s e . The re s ulting ne w, nonre ducing e nd (s e e “j” in Figure 11.5 ), a s we ll a s the old nonre ducing e nd from which the s ix to e ight re s idue s we re re move d (s e e “o” in Figure 11.5), ca n now be furthe r e longa te d by glycoge n s yntha s e . 2. S ynthe s is o f additio nal branc he s : Afte r e longa tion of the s e two

O OH

1. S ynthe s is o f branc he s : Bra nche s a re ma de by the a ction of the

O P O3 OH

Gluc o s e 1-P

A. S ho rte ning o f c hains + HO

HO

HO

O OH

O OH

O OH

HO O

O OH

OH

OH OH

Re maining g lyc o g e n

Fig ure 11.7 Cle a va ge of a n α (1 → 4 )-glycos idic bond. P LP = pyridoxa l phos pha te ; P i = inorga nic phos pha te ; P = phos pha te .

Glycoge n phos phoryla s e s e que ntia lly cle a ve s the α (1→4) glycos idic bonds be twe e n the glucos yl re s idue s a t the nonre ducing e nds of the glycoge n cha ins by s imple phos phorolys is (producing glucos e 1-phos pha te ) until four glucos yl units re ma in on e a ch cha in be fore a bra nch point (Figure 11.7). [Note : P hos phoryla s e conta ins a mole cule of cova le ntly bound pyridoxa l phos pha te tha t is re quire d a s a coe nzyme .] The re s ulting s tructure is ca lle d a limit de xtrin, a nd phos phoryla s e ca nnot de gra de it a ny furthe r (Figure 11.8). B. Re mo val o f branc he s Bra nche s a re re move d by the two e nzymic a ctivitie s of a s ingle bifunctiona l prote in, the de bra nching e nzyme (s e e Figure 11.8). Firs t, oligo-α (1→4)→α (1→4)-gluca ntra ns fe ra s e a ctivity re move s the oute r thre e of the four glucos yl re s idue s a tta che d a t a bra nch. It ne xt

IV. De gra da tion of Glycoge n (Glycoge nolys is )

129

GLYCOGENIN H2 O

α-1,6-bo nd

GLUCOS E Lys os oma l α (1 →4)-glucos ida s e

TYPE II: POMPE DIS EAS E (LYS OS OMAL α (1→4)-GLUCOS IDAS E DEFICIENCY)

NONREDUCING ENDS

TYPE V: Mc ARDLE S YNDROME (S KELETAL MUS CLE GLYCOGEN P HOS P HORYLAS E OR MYOP HOS P HORYLAS E DEFICIENCY) S ke le tal mus c le affe c te d; live r e nzyme no rmal

• mpo rary we akne s s and c ramping o f • Te s ke le tal mus c le afte r e xe rc is e • No ris e in blo o d lac tate during s tre nuo us e xe rc is e • No rmal me ntal de ve lo pme nt • Myo g lo bine mia and myo g lo binuria may be s e e n • Re lative ly be nig n, c hro nic c o nditio n • Hig h le ve l o f g lyc o g e n with no rmal s truc ture in mus c le • De fic ie nc y o f the live r is o zyme c aus e s Type VI:

• Lys o s o mal s to rag e dis e as e • Ge ne ralize d (but primarily he art, live r, mus c le ) e s s ive g lyc o g e n c o nc e ntratio ns • Exc fo und in abno rmal vac uo le s in the lys o s o me s • No rmal blo o d s ug ar le ve ls • Mas s ive c ardio me g aly • Enzyme re plac e me nt the rapy available fo rm: e arly de ath typic ally fro m • Infantile he art failure • No rmal g lyc o g e n s truc ture

Pi

Glycoge n phos phoryla s e

1

Gluc o s e 1-P

He rs dis e as e with mild fas ting hypo g lyc e mia.

• Fas ting hypo g lyc e mia • Abno rmal g lyc o g e n s truc ture with

d e

f

TYPE III: CORI DIS EAS E (4:4 TRANS FERAS E and/o r 1:6 GLUCOS IDAS E DEFICIENCY)

g

fo ur o r o ne g luc o s yl re s idue s at branc h po ints

c b c'

a

LIMIT DEXTRIN

a'

a

f'

e'

d'

b

c

d

e

f

2

g

a'

b'

c'

' e' f

d'

c

d

e

f

g

DEBRANCHING NCHING ENZYME (1:6 g lu c o s id a s e ac tivity)

3

Gluc o s e

a'

b'

c'

d'

e'

f'

DEBRANCHING HING ENZYME (4:4 tra n s fee ra s e ac tivity)

g'

H2 O

a b

b'

g'

Co ntinue d o n ne xt pag e

Fig ure 11.8 Glycoge n de gra da tion, s howing s ome of the glycoge n s tora ge dis e a s e s (GS Ds ). [Note : A GS D ca n a ls o be ca us e d by de fe cts in bra nching e nzyme , a n e nzyme of s ynthe s is , re s ulting in Type IV: Ande rs e n dis e a s e a nd ca us ing de a th in e a rly childhood from live r cirrhos is .](Continue d on ne xt pa ge .) P i = inorga nic phos pha te ; P = phos pha te .

130

11. Glycoge n Me ta bolis m

TYPE Ia: VON GIERKE DIS EAS E (GLUCOS E 6-P HOS P HATAS E DEFICIENCY) TYPE Ib: GLUCOS E 6-PHOS PHATE TRANS LOCAS E DEFICIENCY

• Affe c ts live r and kidne y • Fas ting hypo g lyc e mia– s e ve re • Fatty live r, he pato - and re no me g aly • Pro g re s s ive re nal dis e as e • Gro wth re tardatio n and de laye d pube rty tic ac ide mia, hype rlipide mia, • Lac and hype ruric e mia rmal g lyc o g e n s truc ture ; inc re as e d • No g lyc o g e n s to re d Ib als o c harac te rize d by ne utro pe nia • Type and re c urre nt infe c tio ns atme nt: No c turnal g as tric infus io ns • Tre o f g luc o s e o r re g ular adminis tratio n o f unc o o ke d c o rns tarc h

(Fig ure 11.8 c o ntinue d) Re pe at s te ps

GLUCOS GLUCO E 1-P

1

+

2

3

GLUCOS E

(Ratio ~8:1) MUS CLE

P hos phoglucomuta s e

Gluc o s e 6-P

H2 O

GLYCOLYS IS

LIVER Glucos e 6-phos pha ta s e

Pi Gluc o s e

GLU GLUCOS E

Fig ure 11.8 (Continue d from the pre vious pa ge .) Glycoge n de gra da tion, s howing s ome of the glycoge n s tora ge dis e a s e s (GS Ds ). tra ns fe rs the m to the nonre ducing e nd of a nothe r cha in, le ngthe ning it a ccordingly. Thus , a n α (1→4) bond is broke n a nd a n α (1→4) bond is ma de , a nd the e nzyme functions a s a 4:4 tra ns fe ra s e . Ne xt, the re ma ining glucos e re s idue a tta che d in a n α (1→6) linka ge is re move d hydrolytica lly by a mylo-α (1→6)-glucos ida s e a ctivity, re le a s ing fre e glucos e . The glucos yl cha in is now a va ila ble a ga in for de gra da tion by glycoge n phos phoryla s e until four glucos yl units in the ne xt bra nch a re re a che d. C. Co nve rs io n o f g luc o s e 1-pho s phate to g luc o s e 6-pho s phate Glucos e 1-phos pha te , produce d by glycoge n phos phoryla s e , is conve rte d in the cytos ol to glucos e 6-phos pha te by phos phoglucomuta s e (s e e Figure 11.6). In the live r, glucos e 6-phos pha te is tra ns porte d into the e ndopla s mic re ticulum (ER) by glucos e 6-phos pha te tra ns loca s e . The re it is conve rte d to glucos e by glucos e 6-phos pha ta s e (the s a me e nzyme us e d in the la s t s te p of glucone oge ne s is ; s e e p. 121). The glucos e the n is tra ns porte d from the ER to the cytos ol. He pa tocyte s re le a s e glycoge n-de rive d glucos e into the blood to he lp ma inta in blood glucos e le ve ls until the glucone oge nic pa thwa y is a ctive ly producing glucos e . [Note : In the mus cle , glucos e 6-phos pha te ca nnot be de phos phoryla te d a nd s e nt into the blood be ca us e of a la ck of glucos e 6-phos pha ta s e . Ins te a d, it e nte rs glycolys is , providing e ne rgy ne e de d for mus cle contra ction.] D. Lys o s o mal de g radatio n o f g lyc o g e n A s ma ll a mount (1%–3%) of glycoge n is continuous ly de gra de d by the lys os oma l e nzyme , α (1→4)-glucos ida s e (a cid ma lta s e ). The purpos e of this pa thwa y is unknown. Howe ve r, a de ficie ncy of this e nzyme ca us e s a ccumula tion of glycoge n in va cuole s in the lys os ome s , re s ulting in the s e rious glycoge n s tora ge dis e a s e (GS D) Type II: P ompe dis e a s e (s e e Figure 11.8). [Note : Type II: P ompe dis e a s e is the only GS D tha t is a lys os oma l s tora ge dis e a s e .]

V. Re gula tion of Glycoge ne s is a nd Glycoge nolys is

131

Lys os oma l s tora ge dis e a s e s a re ge ne tic dis orde rs cha ra cte rize d by the a ccumula tion of a bnorma l a mounts of ca rbohydra te s or lipids prima rily due to the ir de cre a s e d lys os oma l de gra da tion.

V. REGULATION OF GLYCOGENES IS AND GLYCOGENOLYS IS Be ca us e of the importa nce of ma inta ining blood glucos e le ve ls , the s ynthe s is a nd de gra da tion of its glycoge n s tora ge form are tightly re gula te d. In the live r, glycoge ne s is a cce le ra te s during pe riods whe n the body ha s be e n we ll fe d, whe re a s glycoge nolys is a cce le ra te s during pe riods of fa s ting. In s ke le ta l mus cle , glycoge nolys is occurs during a ctive e xe rcis e , a nd glycoge ne s is be gins a s s oon a s the mus cle is a ga in a t re s t. Regula tion of glycoge n s ynthe s is a nd de gra da tion is a ccomplis he d on two le ve ls . Firs t, glycoge n s yntha s e a nd glycoge n phos phoryla s e a re hormona lly re gula te d (by phos phoryla tion/de phos phoryla tion) to me e t the ne e ds of the body a s a whole . [Note : P hos phoryla tion of glycoge n phos phoryla s e is ca ta lyze d by glycoge n phos phoryla s e kina s e (s e e p. 132).] S e cond, the s e s a me e nzyme s a re a llos te rica lly re gula te d (by e ffe ctor mole cule s ) to me e t the ne e ds of a pa rticula r tis s ue . Gluc ag o n bo und to g luc ag o n re c e pto r

Epine phrine bo und to β-adre ne rg ic re c e pto r

(LIVER)

(MUS CLE and LIVER)

GTP

GTP

α

Active ade nylyl cycla s e

ROLE OF CALCIUM IN MUS CLE During mus c le c o ntrac tio n, Ca 2 + is re le as e d fro m the s arc o plas mic re tic ulum. Ca 2 + binds to the c almo dulin s ubunit o f p h o s p h o ryla s e kin a s e b , ac tivating it witho ut phos phorylation. Phos phorylas e kinas e can then activate glycogen p h o s p h o ryla s e , c aus ing g lyc o g e n de g radatio n.

ATP A

α

β γ

PP i P hos phodie s te ra s e c AMP ( )

cAMP -de pe nde nt prote iin kina s e A

cAMP -de pe nde nt prote in kina s e A

(in (inac tive )

(ac tive )

R

C

R

C

5'-AMP

+

GLYCOGEN IS DEGRADED

R R

C ATP

P Glycoge n phos phoryla s e a

ADP

P

Glycoge n phos phoryla s e kina s e b

Glycoge n phos phoryla s e kina s e a

(inac tive )

(ac tive ) Pi

ADP

ATP

H2 O

+

P rote in phos pha ta s e -1

(ac tive )

Ins ulin

H2 O P rote in phos pha ta s e -1

Glycoge n phos phoryla s e b

Pi

+

β γ

(inac tive ) Ins ulin

ROLE OF AMP IN MUS CLE In mus c le unde r e xtre me c o nditio ns o f ano xia and de ple tio n o f ATP, AMP ac tivate s g lyc o g e n p h o s p h o ryla s e b witho ut it be ing pho s pho rylate d.

Fig ure 11.9 S timula tion a nd inhibition of glycoge n de gra da tion. AMP = a de nos ine monophos pha te ; cAMP = cyclic AMP ; GTP = gua nos ine triphos pha te ; P = phos pha te ; P P i = pyrophos pha te ; R = re gula tory s ubunit; C = ca ta lytic s ubunit.

132

11. Glycoge n Me ta bolis m A. Ac tivatio n o f g lyc o g e n de g radatio n

Gluc ag o n Epine phrine

(LIVER)

The binding of hormone s , s uch a s gluca gon or e pine phrine , to pla s ma me mbra ne G prote in–couple d re ce ptors (GP CRs ) s igna ls the ne e d for glycoge n to be de gra de d, e ithe r to e le va te blood glucos e le ve ls or to provide e ne rgy for e xe rcis ing mus cle .

(MUS CLE and LIVER)

1. Ac tivatio n o f pro te in kinas e A: Binding of gluca gon or e piAc tive a d e n ylyl c yc la s e

PP i

ATP

P hos pho-

c AMP ( )

die s te ra s e

5'-AMP

cAMP -de pe nde nt prote in kina s e A

(inac tive ) R

C

R

C

ne phrine to the ir s pe cific he pa tocyte GP CR, or of e pine phrine to a s pe cific myocyte GP CR, re s ults in the G prote in–me dia te d a ctiva tion of a de nylyl cycla s e . This e nzyme ca ta lyze s the s ynthe s is of cyclic a de nos ine monophos pha te (cAMP ), which a ctiva te s cAMP -de pe nde nt prote in kina s e A (P KA), a s de s cribe d on pa ge 95. P KA is a te tra me r, ha ving two re gula tory s ubunits (R) a nd two ca ta lytic s ubunits (C). cAMP binds to the re gula tory s ubunits , re le a s ing individua l ca ta lytic s ubunits tha t a re a ctive (Figure 11.9). P KA the n phos phoryla te s s e ve ra l e nzyme s of glycoge n me ta bolis m, a ffe cting the ir a ctivity. [Note : Whe n cAMP is re move d, the ina ctive te tra me r, R 2 C 2 , is a ga in forme d.] 2. Ac tivatio n o f pho s pho rylas e kinas e : P hos phoryla s e kina s e

cAMP -de pe nde nt prote in kina s e A

+

(ac tive )

e xis ts in two forms : a n ina ctive “b” form a nd a n a ctive “a ” form. Active P KA phos phoryla te s the ina ctive “b” form of phos phoryla s e kina s e , producing the a ctive “a ” form (s e e Figure 11.9).

R R

C ATP

3. Ac tivatio n o f g lyc o g e n pho s pho rylas e : Glycoge n phos phoryla s e ADP P

Glycoge n s yntha s e a

Glycoge n s yntha s e b

(ac tive )

(inac tive ) H2 O

Pi

P rote in phos pha ta s e -1

+ Ins ulin

GLYCOGEN S YNTHES IS IS INHIBITED Fig ure 11.10 Hormona l re gula tion of glycoge n s ynthe s is . [Note : In contra s t to glycoge n phos phoryla s e , glycoge n s yntha s e is ina ctiva te d by phos phoryla tion.] cAMP = cyclic a de nos ine monophos pha te ; P = phos pha te ; P P i = pyrophos pha te ; R = re gula tory s ubunit; C = ca ta lytic s ubunit.

a ls o e xis ts in two forms : the de phos phoryla te d, ina ctive “b” form a nd the phos phoryla te d, a ctive “a ” form. Active phos phoryla s e kina s e is the only e nzyme tha t phos phoryla te s glycoge n phos phoryla s e b to its a ctive “a ” form, which the n be gins glycoge nolys is (s e e Figure 11.9). 4. S ummary o f the re g ulatio n o f g lyc o g e n de g radatio n: The ca s -

ca de of re a ctions lis te d a bove re s ults in glycoge nolys is . The la rge numbe r of s e que ntia l s te ps s e rve s to a mplify the e ffe ct of the hormona l s igna l (tha t is , a fe w hormone mole cule s binding to the ir re ce ptors re s ults in a numbe r of P KA mole cule s be ing a ctiva te d tha t ca n e a ch a ctiva te ma ny phos phoryla s e kina s e mole cule s ). This ca us e s the production of ma ny a ctive glycoge n phos phoryla s e a mole cule s tha t ca n de gra de glycoge n. 5. Mainte nance of the phos phorylate d s tate : The phosphate groups

a dde d to phos phoryla s e kina s e a nd phos phoryla s e in re s pons e to cAMP a re ma inta ine d be ca us e the e nzyme tha t hydrolytica lly re move s the phos pha te , prote in phos pha ta s e -1 (P P 1), is ina ctiva te d by inhibitor prote ins tha t a re a ls o phos phoryla te d a nd a ctivate d in re sponse to cAMP (see Figure 11.9). [Note: PP1 is a ctiva ted by a signal cascade initiated by insulin (see p. 311). Insulin a ls o a ctiva te s the phos phodie s te ra s e tha t de gra de s cAMP , a nd, thus, insulin opposes the effects of glucagon and epinephrine.] B. Inhibitio n o f g lyc o g e n s ynthe s is The re gula te d e nzyme in glycoge ne s is is glycoge n s yntha s e . It a ls o e xis ts in two forms , the a ctive “a ” form a nd the ina ctive “b” form. Howe ve r, for glycoge n s yntha s e , in contra s t to phos phoryla s e kina s e a nd phos phoryla s e , the a ctive form is de phos phoryla te d, whe re a s

V. Re gula tion of Glycoge ne s is a nd Glycoge nolys is the ina ctive form is phos phoryla te d (Figure 11.10). Glycoge n s yntha s e a is conve rte d to the ina ctive “b” form by phos phoryla tion a t s e ve ra l s ite s on the e nzyme , with the le ve l of ina ctiva tion proportiona l to its de gre e of phos phoryla tion. P hos phoryla tion is ca ta lyze d by s e ve ra l diffe re nt prote in kina s e s tha t a re re gula te d by cAMP or othe r s igna ling me cha nis ms (s e e C. be low). Glycoge n s yntha s e b ca n be re conve rte d to the “a ” form by P P 1. Figure 11.11 s umma rize s the cova le nt re gula tion of glycoge n me ta bolis m. C. Allo s te ric re g ulatio n o f g lyc o g e n s ynthe s is and de g radatio n In a ddition to hormona l s igna ls , glycoge n s yntha s e a nd glycoge n phos phoryla s e re s pond to the le ve ls of me ta bolite s a nd e ne rgy ne e ds of the ce ll. Glycoge ne s is is s timula te d whe n s ubs tra te a va ila bility a nd e ne rgy le ve ls a re high, whe re a s glycoge nolys is is incre a s e d whe n glucos e a nd e ne rgy le ve ls a re low. This a llos te ric re gula tion a llows a ra pid re s pons e to the ne e ds of a ce ll a nd ca n ove rride the e ffe cts of hormone -me dia te d cova le nt re gula tion. 1. Re g ulatio n o f g lyc o g e n s ynthe s is and de g radatio n in the we llfe d s tate :

2. Ac tivatio n o f g lyc o g e n de g radatio n by c alc ium: Ca 2+ is re le a s e d

mus cle contra ction, the re is a ra pid a nd urge nt ne e d for ATP . This e ne rgy is s upplie d by the de gra da tion of mus cle glycoge n to glucos e , which ca n the n e nte r glycolys is . Ne rve impuls e s ca us e me mbra ne de pola riza tion, which promote s Ca 2+ re le a s e from the s a rcopla s mic re ticulum into the s a rcopla s m of myocyte s . The Ca 2+ binds the Ca M s ubunit, a nd the comple x a ctiva te s mus cle phos phoryla s e kina s e b (s e e Figure 11.9).

c AMP-me diate d ac tivatio n o f P KA and pho s pho rylatio n o f

Glyc o g e n s yn th a s e (inac tive ) (De cre a s e d glycoge ne s is )

P ro te in p h o s p h a ta s e -1 inhibito r (ac tive ) (Ma inte na nce of phos phoryla tion)

P h os p h o ryla s e kin a s e (ac tive ) Pho s pho rylate s P h o s p h o ryla s e (ac tive ) (Incre a s e d glycoge nolys is )

Fig ure 11.11 S umma ry of the hormone -me dia te d cova le nt re gula tion of glycoge n me ta bolis m. cAMP = cyclic AMP ; P KA = prote in kina s e A.

A LIVER Glyc o g e n Gluc o s e 6-P

Gluc o s e 6-P

ATP Glycoge n phos phoryla s e

Glycoge n s yntha s e

Gluc o s e

Gluc o s e 1-pho s phate

B MUS CLE Glyc o g e n Gluc o s e 6-P

Gluc o s e 6-P

ATP Glycoge n phos phoryla s e

AMP

Glycoge n s yntha s e

+

a. Calc ium ac tivatio n o f mus c le pho s pho rylas e kinas e : During

Epine phrine (LIVER + MUS CLE)

+

into the cytopla s m in mus cle in re s pons e to ne ura l s timula tion a nd in live r in re s pons e to e pine phrine binding to α 1-a dre ne rgic re ce ptors . The Ca 2+ binds to ca lmodulin (Ca M), the mos t wide ly dis tribute d me mbe r of a fa mily of s ma ll, ca lcium-binding prote ins . The binding of four mole cule s of Ca 2+ to Ca M trigge rs a conforma tiona l cha nge s uch tha t the a ctiva te d Ca 2+–Ca M comple x binds to a nd a ctiva te s prote in mole cule s , ofte n e nzyme s , that a re ina ctive in the a bs e nce of this comple x (s e e Figure 11.13). Thus , Ca M functions a s a n e s s e ntia l s ubunit of ma ny comple x prote ins . One s uch prote in is the te tra me ric phos phoryla s e kina s e , whos e b form is a ctiva te d by the binding of Ca 2+ to its δ s ubunit (Ca M) without the ne e d for the kina s e to be phos phoryla te d by P KA. [Note : Epine phrine a t β-a dre ne rgic re ce ptors s igna ls through a ris e in cAMP , not Ca 2+ (s e e p. 131).]

Gluc ag o n (LIVER)

+

In the we ll-fe d s ta te , glycoge n s yntha s e b in both live r a nd mus cle is a llos te rica lly a ctiva te d by glucos e 6-phos pha te , which is pre s e nt in e le va te d conce ntra tions (Figure 11.12). In contra s t, glycoge n phos phoryla s e a is a llos te rica lly inhibite d by glucos e 6-phos pha te , a s we ll a s by ATP , a high-e ne rgy s igna l in the ce ll. [Note : In live r, but not mus cle , nonphos phoryla te d glucos e is a ls o a n a llos te ric inhibitor of glycoge n phos phoryla s e a , ma king it a be tte r s ubs tra te for P P 1.]

133

Gluc o s e 1-pho s phate Fig ure 11.12 Allos te ric re gula tion of glycoge n s ynthe s is a nd de gra da tion. A. Live r. B. Mus cle . P = phos pha te .

134

11. Glycoge n Me ta bolis m b. Calc ium ac tivatio n o f live r pho s pho rylas e kinas e : During Ca 2 +

Endo plas mic re tic ulum

Ca 2 + Ca 2 +

Ca 2 + is re le as e d fro m the e ndo plas mic re tic ulum in re s po ns e to ho rmo ne s o r ne uro trans mitte rs binding to c e ll-s urfac e re c e pto rs .

phys iologic s tre s s , e pine phrine is re le a s e d from the a dre na l me dulla a nd s igna ls the ne e d for blood glucos e . This glucos e initia lly come s from he pa tic glycoge nolys is . Binding of epine phrine to he pa tocyte α -a dre ne rgic GP CRs a ctiva te s a phos pholipid-de pe nde nt ca s ca de (s e e p. 205) tha t re s ults in move me nt of Ca 2+ from the ER into the cytopla s m. A Ca 2+–Ca M comple x forms a nd a ctiva te s he pa tic phos phoryla s e kina se b. [Note : The re le a s e d Ca 2+ a ls o he lps to a ctiva te prote in kina s e C tha t ca n phos phoryla te (the re fore , ina ctiva te ) glycoge n s yntha s e a.] 3. Ac tivatio n o f g lyc o g e n de g radatio n in mus c le : Mus cle glycoge n

Ca 2 +

Calmo dulin (CaM)

Ca 2 +

Ca 2 +

Ca 2 + CaM–Ca co mple plexx

Ca 2 +

Ca 2 +

phos phoryla s e is a ctive in the pre s e nce of the high a de nos ine monophos pha te (AMP ) conce ntra tions tha t occur unde r e xtre me conditions of a noxia a nd ATP de ple tion. AMP binds to glycoge n phos phoryla s e b, ca us ing its a ctiva tion without phos phoryla tion (s e e Figure 11.9). [Note : Re ca ll tha t AMP a ls o a ctiva te s phos phofructokina s e -1 of glycolys is (s e e p. 99), a llowing glucos e from glycoge nolys is to be oxidize d.]

VI. GLYCOGEN S TORAGE DIS EAS ES The trans ie nt inc re as e in the intrac e llular Ca 2 + c o nc e ntratio n favo rs the fo rmatio n o f the CaM–Ca 2 +c o mple x.

Inac tive e nzyme

Ca 2 +

CaM–Ca 2 +

Ca 2 +

comple p x

The s e a re a group of ge ne tic dis e a s e s tha t a re ca us e d by de fe cts in e nzyme s re quire d for glycoge n de gra da tion or, more ra re ly, glycoge n s ynthe s is . The y re s ult e ithe r in forma tion of glycoge n tha t ha s a n a bnorma l s tructure or in the a ccumula tion of e xce s s ive a mounts of norma l glycoge n in s pe cific tis s ue s a s a re s ult of impa ire d de gra da tion. A pa rticula r e nzyme ma y be de fe ctive in a s ingle tis s ue , s uch a s live r (re s ulting in hypoglyce mia ) or mus cle (ca us ing mus cle we a kne s s ), or the de fe ct ma y be more ge ne ra lize d, a ffe cting a va rie ty of tis s ue s . The s e ve rity of the GS Ds ra nge s from fa ta l in e a rly childhood to mild dis orde rs tha t a re not life thre a te ning. S ome of the more pre va le nt GS Ds a re illus tra te d in Figure 11.8. [Note : Only one GS D is lys os oma l be ca us e glycoge n me ta bolis m occurs prima rily in the cytos ol.]

Ca 2 +

Ca 2 +

Active Ac tive e nzyme

S ubs trate

Pro duc t

The CaM–Ca 2 +c o mple x is an e s s e ntial c o mpo ne nt o f many Ca 2 +-de pe nde nt e nzyme s .

Fig ure 11.13

Ca lmodulin me dia te s ma ny e ffe cts of intra ce llula r Ca 2+.

VII. CHAPTER S UMMARY The main s tores of glycogen in the body are found in s keletal mus cle, whe re the y s e rve a s a fue l re s e rve for the s ynthe s is of ATP during mus cle contraction, and in the liver, where they are used to maintain the blo o d g luc o s e conce ntra tion, pa rticula rly during the e arly s tag e s of a fas t. Glycogen is a highly branched polymer of α-D-glucos e. The primary glycosidic bond is an α(1→4) linkage. After about eight to ten glucosyl residues, there is a branch containing an α(1→6) linkage. Uridine diphos phate (UDP)-glucos e, the building block of glycogen, is synthesized from glucos e 1-phos phate and UTP by UDP-glucos e pyrophos phorylas e (Figure 11.14). Glucose from UDP-glucose is transferred to the nonreducing ends of glycogen chains by primer-requiring glycogen s ynthas e, which makes α(1→4) linkages . The primer is made by glycogenin. Branc he s a re forme d by a m ylo -α(1→4)→α(1→6)-tra n s g lu c o s id a s e (common name, glucos yl 4:6 trans feras e), which transfers a set of six to eight glucosyl residues from the nonreducing end of the glycogen chain (breaking an α(1→4) linkage), and attaches it with an α(1→6) linkage

VII. Cha pte r S umma ry

135

to another residue in the chain. Pyridoxal phosphate–requiring glycogen phos phorylas e cleaves the α(1→4) bonds between glucosyl residues at the nonreducing ends of the glycogen chains, producing glucos e 1-phos phate. This sequential degradation continues until four glucosyl units remain before a branch point. The resulting structure is called a limit dextrin that is degraded by the bifunctional debranching enzyme. Oligo-α(1→4)→α(1→4)-glucantrans feras e (common name, glucos yl 4:4 trans feras e) removes the outer three of the four glucosyl residues at a branch and transfers them to the nonreducing end of another chain, where they can be converted to glucose 1-phosphate by glycogen phosphorylase. The remaining single glucose residue attached in an α(1→6) linkage is removed hydrolytically by the amylo-(1→6) glucos idas e activity of debranching enzyme, releasing free glucos e. Glucos e 1-phos phate is converted to glucos e 6-phos phate by phos phoglucomutas e. In the mus cle, glucose 6-phosphate enters glycolysis. In the liver, the phosphate is removed by glucos e 6-phos phatas e, releasing free glucos e that can be used to maintain blood glucose levels at the beginning of a fast. A deficiency of the phos phatas e causes glycogen s torage dis eas e Type 1a (Von Gierke dis eas e). This disease results in an inability of the liver to provide free glucose to the body during a fast. It affects both glycogen degradation and gluconeogenesis. Glycogen synthesis and degradation are reciprocally regulated to meet whole-body needs by the same hormonal signals (namely, an elevated ins ulin level results in overall increas ed glycogenes is and decreas ed glycogenolys is , whereas an elevated glucagon, or epinephrine, level causes increas ed glycogenolys is and decreas ed glycogenes is ). Key enzymes are phosphorylated by a family of protein kinas es , some of which are cyclic adenos ine monophos phate dependent (a compound increased by glucagon and epinephrine). Phosphate groups are removed by protein phosphatase-1 (active when its inhibitor is inactive in response to elevated insulin levels). Glycogen s ynthas e, phos phorylas e kinas e, and phos phorylas e are also allos terically regulated to meet tissues needs. In the well-fed state, glycogen s ynthas e is activated by glucos e 6-phos phate, but glycogen phos phorylas e is inhibited by glucos e 6-phos phate as well as by ATP. In the liver, glucose also serves an an allosteric inhibitor of glycogen phosphorylase. The Ca 2+ released from the endoplasmic reticulum in muscle during exercise and in liver in response to epinephrine activates phos phorylas e kinas e by binding to the enzyme’s calmodulin subunit. This allows the enzyme to activate glycogen phos phorylas e, thereby causing glycogen degradation. AMP activates glycogen phosphorylase in muscle.

Me ta b o li c c h a ra c te ris ti c s

LIVER, MUS CLE

occurs ma inly in

Cytos ol

occurs in

R e g u l a ti o n

Re gula te d e nzyme s

Glyc o g e n UDP-Gluc o s e

Glucos e 6-P

re quire s

UTP

Gluc o s e 1-P Glycoge n s yntha s e

We ll-fe d s tate

Fas ting s tate Glycoge n phos phoryla s e

Inge s tion of glucos e

Inge s tion of food

le a ds to

le a ds to Blood glucos e

le a ds to

Re le a s e of ins ulin

le a ds to de phos phoryla tion

le a ds to le a ds to phos phoryla tion

ATP Glucos e 6-P

Re le a s e of ins ulin

AMP (mus cle )

le a ds to P rote in phos pha ta s e a ctivity

Conve rs ion of glycoge n to glucos e

Blood glucos e

le a ds to

Glucos e (live r)

Re le a s e of gluca gon

le a ds to

Conve rs ion of glycoge n to glucos e

Re le a s e of gluca gon

le a ds to P rote in kina s e a ctivity

Fig ure 11.14 Ke y conce pt ma p for glycoge n me ta bolis m in the live r. [Note : Glycoge n phos phoryla s e is phos phoryla te d by phos phoryla s e kina s e , the “b” form of which ca n be a ctiva te d by ca lcium.] UDP = uridine diphos pha te ; UTP = uridine triphos pha te ; P = phos pha te .

136

11. Glycoge n Me ta bolis m

S tudy Que s tio ns Choos e the ONE be s t a ns we r. For Que s tions 11.1–11.4, ma tch the de ficie nt e nzyme to the clinica l finding in s e le cte d glycoge n s tora ge dis e a s e s (GS Ds ). CHOICE

GS D

DEFICIENT ENz y ME

A

Type Ia

Glucos e 6-phos pha ta s e

B

Type II

Acid ma lta s e

C

Type III

4:4 Tra ns fe ra s e

D

Type IV

4:6 Tra ns fe ra s e

E

Type V

Myophos phoryla s e

F

Type VI

Live r phos phoryla s e

11.1 Exe rcise intole ra nce , with no rise in blood la cta te during e xe rcis e 11.2 Fa ta l, progre s s ive cirrhos is a nd glycoge n with longe rtha n-norma l oute r cha ins

Corre ct a ns we r = E. Myophos phoryla s e de ficie ncy pre ve nts glycoge n de gra da tion in mus cle , de priving mus cle of glycoge n-de rive d glucos e , re s ulting in de cre a s e d glycolys is a nd its a na e robic product, la cta te . Corre ct Ans we r = D. 4:6 Tra ns fe ra s e (bra nching e nzyme ) de ficie ncy, a de fe ct in glycoge n s ynthe s is , re s ults in forma tion of glycoge n with fe we r bra nche s a nd de cre a s e d s olubility. Corre ct a ns we r = B. Acid ma lta s e [α (1→4)glucos ida s e ] de ficie ncy pre ve nts de gra da tion of a ny glycoge n brought into lys os ome s . A va rie ty of tis s ue s a re a ffe cte d, with the mos t s e ve re pa thology re s ulting from he a rt da ma ge . Corre ct a ns we r = A. Glucos e 6-phos pha ta s e de ficie ncy pre ve nts the live r from re le a s ing fre e glucos e into the blood, ca us ing s e ve re fa s ting hypoglyce mia , la ctica cide mia , hype rurice mia , a nd hype rlipide mia .

11.3 Ge ne ra lize d a ccumula tion of glycoge n, s e ve re hypotonia , a nd de a th from he a rt fa ilure 11.4 S e ve re fa s ting hypoglyce mia , la ctica cide mia , hype rurice mia , a nd hype rlipide mia 11.5 Epine phrine a nd gluca gon ha ve which one of the following e ffe cts on he pa tic glycoge n me ta bolis m? A. Both glycoge n phos phoryla s e a nd glycoge n s yntha s e a re a ctiva te d by phos phoryla tion but a t s ignifica ntly diffe re nt ra te s . B. Glycoge n phos phoryla s e is ina ctiva te d by the re s uting ris e in ca lcium, whe re a s glycoge n s yntha s e is a ctiva te d. C. Glycoge n phos phoryla s e is phos phoryla te d a nd a ctive , whe re a s glycoge n s yntha s e is phos phoryla te d a nd ina ctive . D. The ne t s ynthe s is of glycoge n is incre a s e d. 11.6 In contra cting s ke le ta l mus cle , a s udde n e le va tion of the s a rcopla s mic ca lcium conce ntra tion will re s ult in: A. a ctiva tion of cyclic a de nos ine monophos pha te (cAMP )-de pe nde nt prote in kina s e A. B. conve rsion of cAMP to AMP by phosphodie ste ra se . C. dire ct a ctiva tion of glycoge n s yntha s e b. D. dire ct a ctiva tion of phos phoryla s e kina s e b. E. ina ctiva tio n of ph os ph oryla s e kin a s e a by th e a ction of prote in phos pha ta s e -1. 11.7 Expla in why the hypoglyce mia s e e n with Type Ia glycoge n s tora ge dis e a s e (glucos e 6-phos pha ta s e de ficie ncy) is s e ve re , whe re a s tha t s e e n with Type VI (live r phos phoryla s e de ficie ncy) is mild.

Corre ct a ns we r = C. Epine phrine a nd gluca gon both ca us e incre a s e d glycoge n de gra da tion a nd de cre a s e d s ynthe s is in the live r through cova le nt modifica tion (phos phoryla tion) of ke y e nzyme s of glycoge n me ta bolis m. Glycoge n phos phoryla s e is phos phoryla te d a nd a ctive (“a ” form), whe re a s glycoge n s yntha s e is phos phoryla te d a nd ina ctive (“b” form). Gluca gon doe s not ca us e a ris e in ca lcium.

Corre ct a ns we r = D. Ca 2+ re le a s e d from the s a rcopla s mic re ticulum during e xe rcis e binds to the ca lmodulin s ubunit of phos phoryla s e kina s e , the re by a llos te rica lly a ctiva ting the “b” form of this e nzyme . The othe r choice s a re not ca us e d by a n e le va tion of cytos olic ca lcium.

With Type Ia , the live r is una ble to ge ne ra te fre e glucos e e ithe r from glycoge nolys is or glucone oge ne s is be ca us e both proce s s e s produce glucos e 6-phos pha te . With Type VI, the live r is s till a ble to produce fre e glucos e from glucone oge ne s is , but glycoge nolys is is inhibite d.

Me tabo lis m o f Mo no s ac c haride s and Dis ac c haride s

12

I. OVERVIEW Glycoge n

Glucos e is the mos t common monos a ccha ride cons ume d by huma ns , a nd its me ta bolis m ha s a lre a dy be e n dis cus s e d. Howe ve r, two othe r monos accha ride s , fructos e a nd ga la ctose , occur in significa nt a mounts in the die t (prima rily in dis a ccha rides ) a nd ma ke important contributions to ene rgy me ta bolis m. In a ddition, ga lactos e is a n importa nt compone nt of structura l ca rbohydra te s . Figure 12.1 shows the me ta bolis m of fructos e and ga lactos e as pa rt of the es s entia l pa thwa ys of ene rgy me ta bolis m.

Ga la ctos e

UDP -Glucos e Glucos e 1-P

About 10% of the ca lorie s compris ing the We s te rn die t a re s upplie d by fructos e (a pproxima te ly 55 g/da y). The ma jor s ource of fructos e is the dis a ccha ride s ucros e , which, whe n cle a ve d in the inte s tine , re le a s e s e quimola r a mounts of fructos e a nd glucos e . Fructos e is a ls o found a s a fre e monos a ccha ride in ma ny fruits , in hone y, a nd in high-fructos e corn s yrup (typica lly, 55% fructos e /45% glucos e ), which is us e d to s we e te n s oft drinks a nd ma ny foods . Fructos e tra ns port into ce lls is not ins ulin de pe nde nt (unlike tha t of glucos e into ce rta in tis s ue s ; s e e p. 97), a nd, in contra s t to glucos e , fructos e doe s not promote the s e cre tion of ins ulin.

UDP -Ga la ctos e

Glucos e 6-P

Glucos e

6-P Glucona te Ribulos e 5-P

II. FRUCTOS E METABOLIS M

Ga la ctos e 1-P

Glycoge n UDP -Glucos e

6-P Gluconola ctone

Ribos e 5-P

Ga la ctos e Ga la ctos e 1-P

Glucos e 1-P

Xylulos e 5-P

Glucos e 6-P

S e dohe ptulos e 7-P Erythros e 4-P

Fructos e 6-P

UDP -Ga la ctos e Glucos e Fructos e

Fructos e 1,6-bis -P Glyce ra lde hyde 3-P

Glyce ra lde hyde 3-P

Glyce ra lde hyde

Fructos e 1-P

Dihydroxya ce tone -P Glyce rol-P

1,3-bis -P hos phoglycera te

Glyce rol

3-P hos phoglyce ra te Tria cylglyce rol 2-P hos phoglyce ra te

Ala Cys Gly Ser Thr Try NH3

Fa tty a cid

Fa tty a cyl CoA P hos phoe nolpyruvate La cta te CO 2

CO 2

P yruva te CO2

Ma lonyl CoA

Ace tyl CoA

Ace toa ce ta te

Le u P he Tyr Trp Lys

As n Ca rba moyl-P

β-Hydroxybutyra te As pa rta te

Citrulline

Ornithine

Oxa loa ce ta te

Citra te

Ma la te

Is ocitra te CO 2 α -Ke togluta ra te

Argininos uccina te Fuma ra te

A. Pho s pho rylatio n o f fruc to s e Arginine

S uccina te

CO 2 S uccinyl CoA

Gln P ro His Arg

Glu Me thylma lonyl CoA

Ure a

For fructos e to e nte r the pa thwa ys of inte rme dia ry me ta bolis m, it mus t firs t be phos phoryla te d (Figure 12.2). This ca n be a ccomplis he d by e ithe r he xokina s e or fructokina s e . He xokina s e phos phoryla te s glucos e in mos t ce lls of the body (s e e p. 98), a nd s e ve ra l a dditiona l he xos e s ca n s e rve a s s ubs tra te s for this e nzyme . Howe ve r, it ha s a low a ffinity (tha t is , a high Micha e lis cons ta nt [Km ]; s e e p. 59) for fructos e . The re fore , unle s s the intra ce llula r conce ntra tion of fructos e be come s unus ua lly high, the norma l pre s e nce of s a tura ting conce ntra tions of glucos e me a ns tha t little fructos e is phos phoryla te d by he xokina s e . Fructokina s e provide s the prima ry me cha nis m for fructos e phos phoryla tion (s e e Figure 12.2). The e nzyme ha s a low Km for fructos e a nd a high Vma x (or, ma xima l ve locity; s e e p. 59). It is found in the live r (which proces s e s mos t of the die ta ry fructos e ), kidne y, a nd the s ma ll inte s tina l mucos a a nd conve rts fructos e to fructos e 1-phos pha te , us ing a de nos ine triphos pha te (ATP ) a s the phos pha te donor. [Note : The s e thre e tis s ue s a ls o conta in a ldola s e B, dis cus s e d in s e ction B.]

Ile Me t Va l Thr

P he Tyr

P ropionyl CoA Ace tyl CoA Fa tty a cyl CoA (odd-numbe r ca rbons )

Fructos e Glyce ra lde hyde Glyce ra lde hyde 3-P

Fructos e 1-P

Dihydroxya ce tone P

Fig ure 12.1 Ga la ctos e a nd fructos e me ta bolis m a s pa rt of the e s s e ntia l pa thwa ys of e ne rgy me ta bolis m (s e e Figure 8.2, p. 92, for a more de ta ile d vie w of the ove ra ll re a ctions of me ta bolis m). UDP = uridine diphos pha te ; P = phos pha te .

137

138

12. Me ta bolis m of Monos a ccha ride s a nd Dis a ccha ride s B. Cle avag e o f fruc to s e 1-pho s phate

FRUCTOS E METABOLIS M

GLYCOLYS IS

Fructos e 1-phos pha te is not phos phoryla te d to fructos e 1,6-bis phos pha te a s is fructos e 6-phos pha te (s e e p. 99) but is cle a ve d by a ldola s e B (a ls o ca lle d fructos e 1-phos pha te a ldola s e ) to dihydroxya ce tone phos pha te (DHAP ) a nd glyce ra lde hyde . [Note : Huma ns e xpre s s thre e a ldola s e s , A, B a nd C, the products of thre e diffe re nt ge ne s . Aldola s e A (found in mos t tis s ue s ), a ldola s e B (in live r, kidne y, a nd s ma ll inte s tine ), a nd a ldola s e C (in bra in) a ll cle a ve fructos e 1,6-bis phos pha te produce d during glycolys is to DHAP a nd glyce ra lde hyde 3-phos pha te (s e e p. 100), but only a ldola s e B cle a ve s fructos e 1-phos pha te .] DHAP ca n dire ctly e nte r glycolys is or glucone oge ne s is , whe re a s glyce ra lde hyde ca n be me ta bolize d by a numbe r of pa thwa ys , a s illus tra te d in Figure 12.3.

Unle s s the intrac e llular c o nc e ntratio n o f fruc to s e be c o me s unus ually hig h, h e xo kin a s e is s aturate d with and pho s pho rylate s g luc o s e rathe r than fruc to s e .

CH2 OH C O HO C H H C OH

CH2 OH C O HO C H H C OH

H C OH

H C OH

Fruc to s e He xokina s e ATP

ATP

Fructokina s e

The ra te of fructos e me ta bolis m is more ra pid tha n tha t of glucos e because the trioses formed from fructose 1-phosphate bypass phosphofructokinase-1, the major rate-limiting step in glycolysis (see p. 99).

P hos phofructokina s e

ADP

CH2 O

C. Kine tic s o f fruc to s e me tabo lis m

CH2 O P Fruc to s e 6-pho s phate

CH2 OH

ADP

CH2 O

P

C O HO C H H C OH

C O HO C H H C OH

H C OH

H C OH CH2 O

CH2 OH

P

P

Fruc to s e 1,6-bis pho s phate

Fruc to s e 1-pho s phate

Aldola s e B

Aldola se A, B, C

O

O

C H

C H H H C OH

H C OH CH2 OH

H C OH CH2 O

C O CH2 O

P

P

Glyc e ralde hyde 3-pho s phate Dihydro xyac e to ne pho s phate

Glyc e ralde hyde

D. Dis o rde rs o f fruc to s e me tabo lis m A de ficie ncy of one of the ke y e nzyme s re quire d for the e ntry of fructos e into me ta bolic pa thwa ys ca n re s ult in e ithe r a be nign condition a s a re s ult of fructokina s e de ficie ncy (e s s e ntia l fructos uria ) or a s e ve re dis turba nce of live r a nd kidne y me ta bolis m a s a re s ult of a ldola s e B de ficie ncy (he re dita ry fructos e intole ra nce [HFI]), which is e s tima te d to occur in 1:20,000 live births (s e e Figure 12.3). The firs t s ymptoms of HFI a ppe a r whe n a ba by is we a ne d from milk (s e e p. 142) a nd be gins to be fe d food conta ining s ucros e or fructos e . Fructos e 1-phos pha te a ccumula te s , re s ulting in a drop in the le ve l of inorga nic phos pha te (P i) a nd, the re fore , of ATP production. As ATP fa lls , a de nos ine monophos pha te (AMP ) ris e s . The AMP is de gra de d, ca us ing hype rurice mia (a nd la ctic a cidos is ; s e e p. 299). The de cre a s e d a va ila bility of he pa tic ATP a ffe cts glucone oge ne s is (ca us ing hypoglyce mia with vomiting) a nd prote in s ynthe s is (ca us ing a de cre a s e in blood clotting fa ctors a nd othe r e s s e ntia l prote ins ). Kidne y function ma y a ls o be a ffe cte d. [Note : The drop in P i a ls o inhibits glycoge nolys is (s e e p. 128).] Dia gnos is of HFI ca n be ma de on the ba s is of fructos e in the urine , e nzyme a s s a y us ing live r ce lls , or by DNA-ba s e d te s ting (s e e Cha pte r 33). Aldola s e B de ficie ncy is pa rt of the ne wborn s cre e ning pa ne l. With HFI, s ucros e , a s we ll a s fructos e , mus t be re move d from the die t to pre ve nt live r fa ilure a nd pos s ible de a th. Individua ls with HFI dis pla y a n a ve rs ion to s we e ts a nd, cons e que ntly, ha ve a n a bs e nce of de nta l ca rie s . E. Co nve rs io n o f manno s e to fruc to s e 6-pho s phate

Fig ure 12.2 P hos phoryla tion products of fructos e a nd the ir cle a va ge . P = phos pha te ; ADP = a de nos ine diphos pha te .

Ma nnos e , the C-2 e pime r of glucos e (s e e p. 84), is a n importa nt compone nt of glycoprote ins (s e e p. 166). He xokina s e phos phoryla te s ma nnos e , producing ma nnos e 6-phos pha te , which, in turn, is re ve rs ibly is ome rize d to fructos e 6-phos pha te by phos phoma nnos e is ome ra s e . [Note : The re is little ma nnos e in die ta ry ca rbohydra te s . Mos t intra ce llula r ma nnos e is s ynthe s ize d from fructos e or is pre e xis ting ma nnos e produce d by the de gra da tion of s tructura l ca rbohydra te s a nd s a lva ge d by he xokina s e .]

II. Fructos e Me ta bolis m

139

ATP

ADP

He xokina s e

GLUCOS E

Glucos e 6-phos pha ta s e P hos phoglucois ome ra s e

Pi

S UCROS E

S ucra s e

ES S ENTIAL FRUCTOS URIA Lac k o f fru c to kin a s e

• Auto s o mal re c e s s ive (1:130,000 • births ) • Be nig n c o nditio n • Fruc to s e ac c umulatio n in urine

GLYCOGEN

Gluc o s e 6-P

FRUCTOS E FRUC

GLUCONEOGENES IS

Fruc to s e 6-P

ATP

Fructos e 1,6bis phos pha ta s e

Fructokina s e

Pi

ADP

Fruc to s e 1,6-bis -P

Fruc to s e 1-P Aldola s e A, B, C Aldola s e B

Dihydro xyac e to ne P

HEREDITARY FRUCTOS E INTOLERANCE

• Auto s o mal re c e s s ive (1:20,000 births ) e nc e o f a ld o la s e B le ads to intrac e llular • Abs trapping o f fruc to s e 1-pho s phate e s s e ve re hypo g lyc e mia, vo miting , • Caus jaundic e , he mo rrhag e , he pato me g aly, • •

re nal dys func tio n, hype ruric e mia, and lac tic ac ide mia Fruc to s e , s uc ro s e , and s o rbito l c an c aus e he patic failure and de ath Tre atme nt is re mo val o f fruc to s e and s uc ro s e fro m the die t

Glyc e ralde hyde NADH + H+ Alcohol de hydroge na s e

Trios e P is ome ra s e

Trios e kina s e

NAD+

Glyc e ralde hyde 3-P Glyc e ro l

ADP Trios e P is ome ra s e

ATP Glyce rol kina s e

Dihydro xyac e to ne P NADH + H+

GLYCOLYS IS

Glyce rol 3-P de hydroge na s e

ADP

PHOS PHOGLYCERIDES

ATP

Glyc e ro l 3-P

NAD+

PYRUVATE

TRIACYLGLYCEROLS Fig ure 12.3 S umma ry of fructos e me ta bolis m. P = phos pha te ; P i = inorga nic phos pha te ; NAD(H) = nicotina mide a de nine dinucle otide ; ADP = a de nos ine diphos pha te . F. Co nve rs io n o f g luc o s e to fruc to s e via s o rbito l Mos t s uga rs a re ra pidly phos phoryla te d following the ir e ntry into ce lls . The re fore , the y a re tra ppe d within the ce lls , be ca us e orga nic phos pha te s ca nnot fre e ly cros s me mbra ne s without s pe cific tra ns porte rs . An a lte rna te me cha nis m for me ta bolizing a monos a ccha ride is to conve rt it to a polyol (s uga r a lcohol) by the re duction of a n a lde hyde group, the re by producing a n a dditiona l hydroxyl group. 1. Synthe s is of s orbitol: Aldose reductase reduces glucose, produc-

ing sorbitol (glucitol; Figure 12.4). This enzyme is found in many tissues, including the lens, retina , Schwann cells of peripheral nerves, liver, kidne y, placenta, re d blood cells, and cells of the ovarie s and se minal ve sicle s. In cells of the liver, ovarie s, and seminal vesicles, the re is a s e cond e nzyme , s orbitol de hydroge na s e , which ca n oxidize the sorbitol to produce fructose (see Figure 12.4). The tworeaction pathway from glucose to fructose in the se minal vesicles benefits spe rm cells, which use fructose a s a major carbohydrate e ne rgy s ource . The pa thwa y from s orbitol to fructose in the live r

140

12. Me ta bolis m of Monos a ccha ride s a nd Dis a ccha ride s

A

S EMINAL VES ICLES Glyc o lys is

CHO H C OH HO C H H C OH H C OH

BLOOD

CH2 OH

Gluc o s e

Gluc o s e

NADPH + H+

Aldos e re ducta s e

NADP +

CH2OH H C OH HO C H H C OH H C OH CH2 OH

S o rbito l NAD+ S orbitol de hydroge na s e

NADH + H+

provide s a mechanism by which any a vailable sorbitol is converted into a substrate that can enter glycolysis or gluconeogenesis. 2. Effe c t o f hype rg lyc e mia o n s o rbito l me tabo lis m: Be ca us e ins ulin is

not re quire d for the e ntry of glucos e into the ce lls lis te d in the pre vious pa ra gra ph, la rge a mounts of glucos e ma y e nte r the s e ce lls during time s of hype rglyce mia (for e xa mple , in uncontrolle d dia be te s ). Ele va te d intra ce llula r glucos e conce ntra tions a nd a n a de qua te s upply of re duce d nicotina mide a de nine dinucle otide phos pha te (NADPH) ca us e a ldos e re ducta s e to produce a significa nt incre a s e in the a mount of s orbitol, which ca nnot pa s s e fficie ntly through ce ll me mbra ne s a nd, in turn, re ma ins tra ppe d ins ide the ce ll (s e e Figure 12.4). This is e xa ce rba te d whe n s orbitol de hydroge na s e is low or a bs e nt (for e xa mple , in re tina , le ns , kidne y, a nd ne rve ce lls ). As a re s ult, s orbitol a ccumula te s in the s e ce lls , ca us ing s trong os motic e ffe cts a nd, the re fore , ce ll s we lling a s a re s ult of wa te r re te ntion. Some of the pa thologic a lte ra tions a s s ocia te d with dia be te s ca n be a ttribute d, in pa rt, to this phe nome non, including ca ta ra ct forma tion, pe riphe ra l ne uropa thy, a nd microva s cula r proble ms le a ding to ne phropa thy a nd re tinopa thy. (S ee p. 344 for a dis cus s ion of the complica tions of dia be te s .) [Note : Us e of NADP H in the a ldos e re ducta s e re a ction de cre a s e s the ge ne ra tion of re duce d gluta thione , a n importa nt a ntioxida nt (s e e p. 148), a nd ma y be re la te d to dia be tic complica tions .]

III. GALACTOS E METABOLIS M

CH2 OH

C O HO C H H C OH H C OH CH2 OH

Fruc to s e

B

LENS ES NERVES KIDNEYS Glyc o lys is

BLOOD

Gluc o s e (e le vate d)

G lu c o s e NADPH + H+ Aldos e re ducta s e

NADP +

S o r b it o l H2 O H2 O H2 O

Fig ure 12.4 S orbitol me ta bolis m. NAD(H) = nicotina mide a de nine dinucle otide ; NADP (H) = nicotina mide a de nine dinucle otide phos pha te .

The ma jor die ta ry s ource of ga la ctos e is la ctos e (ga la ctos yl β-1,4glucos e ) obta ine d from milk a nd milk products . [Note : The dige s tion of la ctos e by β-ga la ctos ida s e (la cta s e ) of the inte s tina l mucos a l ce ll me mbra ne wa s dis cus s e d on p. 87.] S ome ga la ctos e ca n a ls o be obta ine d by lys os oma l de gra da tion of comple x ca rbohydra te s , s uch a s glycoprote ins a nd glycolipids , which a re importa nt me mbra ne compone nts . Like fructos e , the tra ns port of ga la ctos e into ce lls is not ins ulin de pe nde nt. A. Pho s pho rylatio n o f g alac to s e Like fructos e , ga la ctos e mus t be phos phoryla te d be fore it ca n be furthe r me ta bolize d. Mos t tis s ue s ha ve a s pe cific e nzyme for this purpos e , ga la ctokina s e , which produce s ga la ctos e 1-phos pha te (Figure 12.5). As with othe r kina s e s , ATP is the phos pha te donor. B. Fo rmatio n o f uridine dipho s phate -g alac to s e Ga la ctos e 1-phos pha te ca nnot e nte r the glycolytic pa thwa y unle s s it is firs t conve rte d to uridine diphos pha te (UDP )-ga la ctos e (Figure 12.6). This occurs in a n e xcha nge re a ction, in which UDP -glucos e re a cts with ga la ctos e 1-phos pha te , producing UDP -ga la ctos e a nd glucos e 1-phos pha te (s e e Figure 12.5). The e nzyme tha t ca ta lyze s this re a ction is ga la ctos e 1-phos pha te uridylyltra ns fe ra s e (GALT). C. Us e o f uridine dipho s phate -g alac to s e as a c arbo n s o urc e fo r g lyc o lys is o r g luc o ne o g e ne s is For UDP -ga la ctos e to e nte r the ma ins tre a m of glucos e me ta bolis m, it mus t firs t be conve rte d to its C-4 e pime r, UDP -glucos e , by UDP -

III. Ga la ctos e Me ta bolis m

141

GALACTOKINAS E DEFICIENCY Rare auto s o mal re c e s s ive dis o rde r

CLAS S IC GALACTOS EMIA Ga la c to s e 1-p h o s p h a te u rid ylytra n s fe ra s e de fic ie nc y

• • Auto s o mal re c e s s ive dis o rde r (1:30,000 births ) e s g alac to s e mia and g alac to s uria, vo miting , • Caus diarrhe a, and jaundic e • Ac c umulatio n o f g alac to s e 1-pho s phate and g alac tito l

• e s e le vatio n o f g alac to s e in • Caus blo o d (g alac to s e mia) and urine (g alac to s uria)

• Caus e s g alac tito l ac c umulatio n if g alac to s e pre s e nt in die t

in ne rve , le ns , live r, and kidne y tis s ue c aus e s live r damag e , s e ve re inte lle c tual dis ability, and c atarac ts

• Ele vate d g alac tito l c an c aus e c atarac ts • Tre atme nt is die tary re s tric tio n Galac tito l

• Pre natal diag no s is po s s ible by c ho rio nic villus s ampling ; ne wbo rn s c re e ning available

• Tre atme nt is re mo val o f g alac to s e (and, the re fo re , lac to s e )

NADP +

fro m die t

s pite ade quate tre atme nt, at ris k fo r de ve lo pme ntal • De de lays and, in fe male s , pre mature o varian failure

NADPH + H+ Aldos e re ducta s e

Glyc o g e n GALACTOS E



ATP

ADP

LACTOS E



UDP-Gluc o s e

Ga la ctos e 1-phos pha te uridylyltra ns fe ra s e

UDP-GALACTOS E

UDP -Glucos e pyrophos phoryla s e

Gluc o s e 1-P UTP P hos phoglucomuta s e

g alac to s e le ve ls are hig h (as in g alac to s e mia). Ele vate d g alac tito l c an c aus e c atarac ts .

Galac to s e 1-P

PP i

ALDOS E REDUCTAS E Pre s e nt in the live r, kidne y, re tina, le ns , ne rve tis s ue , s e minal ve s ic le s , and o varie s .

io lo g ic ally unimpo rtant • inPhys g alac to s e me tabo lis m unle s s

Ga la ctokina s e

UDP -He xos e 4-e pime ra s e

GLYCOLIPIDS GLYCOPROTEINS GLYCOS AMINOGLYCANS

Gluc o s e 6-P Glucos e 6-phos pha ta s e (live r)

UDP-GLUCOS E

GLYCOLYS IS

GLUCOS E

Fig ure 12.5 Me ta bolis m of ga la ctos e . UDP = uridine diphos pha te ; UTP = uridine triphos pha te ; P = phos pha te ; P P i = pyrophos pha te ; NADP (H) = nicotina mide a de nine dinucle otide phos pha te ; ADP = a de nos ine diphos pha te . he xos e 4-e pime ra s e . This “ne w” UDP -glucos e (produce d from the origina l UDP -ga la ctos e ) ca n the n pa rticipa te in ma ny bios ynthe tic re a ctions a s we ll a s be ing us e d in the GALT re a ction de s cribe d a bove . (S e e Figure 12.5 for a s umma ry of this inte rconve rs ion.) D. Ro le o f uridine dipho s phate -g alac to s e in bio s ynthe tic re ac tio ns UDP -ga la ctos e ca n s e rve a s the donor of ga la ctos e units in a numbe r of s ynthe tic pa thwa ys , including s ynthe s is of la ctos e (s e e be low), glycoprote ins (s e e p. 166), glycolipids (s e e p. 210), a nd glycos a minoglyca ns (s e e p. 158). [Note : If ga la ctos e is not provide d by the die t (for e xa mple , whe n it ca nnot be re le a s e d from la ctos e a s a re s ult of a la ck of β-ga la ctos ida s e in pe ople who a re la ctos e intole ra nt), a ll tis s ue re quire me nts for UDP -ga la ctos e ca n be me t by the a ction of UDP -he xos e 4-e pime ra s e on UDP -glucos e , which is e fficie ntly produce d from glucos e 1-phos pha te (s e e Figure 12.5).]

UDP-Galac to s e O HOCH2 HO H

HN O

H OH H

CH

O C CH N O O H O P O P O CH2 O OOH O H H H H

E. Dis o rde rs o f g alac to s e me tabo lis m GALT is de ficie nt in individua ls with cla s s ic ga la ctos e mia (s e e Figure 12.5). In this dis orde r, ga la ctos e 1-phos pha te a nd, the re fore , ga la ctos e a ccumula te . P hys iologic cons e que nce s a re s imila r to thos e found in he re dita ry fructos e intole ra nce (s e e p. 138), but a broa de r s pe ctrum of tis s ue s is a ffe cte d. The a ccumula te d ga la ctos e is s hunte d into s ide pa thwa ys s uch a s tha t of ga la ctitol production.

H

C

HO

Galac to s e

OH

UDP

Fig ure 12.6 S tructure of UDP -ga la ctos e . UDP = uridine diphos pha te .

142

12. Me ta bolis m of Monos a ccha ride s a nd Dis a ccha ride s This re a ction is ca ta lyze d by a ldos e re ducta s e , the s a me e nzyme tha t conve rts glucos e to s orbitol (s e e p. 139). Tre a tme nt re quire s re mova l of ga la ctos e a nd la ctos e from the die t. GALT de ficie ncy is pa rt of the ne wborn s cre e ning pa ne l. [Note : A de ficie ncy in ga la ctokina s e re s ults in a le s s s e ve re dis orde r of ga la ctos e mia me ta bolis m, a lthough ca ta ra cts a re common (s e e Figure 12.5).]

β-D-Ga la c to s yltra n s fe ra s e (pro te in A)

α-Lac talbumin (pro te in B)

IV. LACTOS E S YNTHES IS

UDP -g a la c to s e :g lu c o s e g a la c to s yltra n s fe ra s e (la c to s e s yn th a s e )

UDP-g alac to s e + g luc o s e

UDP

Lac to s e

HO

CH2 OH O

CH2 OH O O

OH

HOH

OH

OH

OH

β-Galac to s e

Gluc o s e

Fig ure 12.7 La ctos e s ynthe s is . UDP = uridine diphos pha te .

La ctos e is a dis a ccha ride tha t cons is ts of a mole cule of β-ga la ctos e a tta che d by a β(1→4) linka ge to glucos e . The re fore , la ctos e is ga la ctos yl β(1→4)-glucos e . La ctos e , known a s “milk s uga r,” is ma de by la cta ting (milk-producing) ma mma ry gla nds . The re fore , milk a nd othe r da iry products a re the die ta ry s ource s of la ctos e . La ctos e is s ynthe s ize d in the Golgi by la ctos e s yntha s e (UDP -ga la ctos e :glucos e ga la ctos yltra ns fe ra s e ), which tra ns fe rs ga la ctos e from UDP -ga la ctos e to glucos e , re le a s ing UDP (Figure 12.7). This e nzyme is compos e d of two prote ins , A a nd B. P rote in A is a β-D-ga la ctos yltra ns fe ra s e a nd is found in a numbe r of body tis s ue s . In tis s ue s othe r tha n the la cta ting ma mma ry gla nd, this e nzyme tra ns fe rs ga la ctos e from UDP -ga la ctos e to N-a ce tylD-glucos a mine , forming the s a me β(1→4) linka ge found in la ctos e , a nd producing N-a ce tylla ctos a mine , a compone nt of the s tructura lly importa nt N-linke d glycoprote ins (s e e p. 167). In contra s t, prote in B is found only in la cta ting ma mma ry gla nds . It is α -la cta lbumin, a nd its s ynthe s is is s timula te d by the pe ptide hormone prola ctin. P rote in B forms a comple x with the e nzyme , prote in A, cha nging the s pe cificity of tha t tra ns fe ra s e s o tha t la ctos e , ra the r tha n N-a ce tylla ctos a mine , is produce d (s e e Figure 12.7).

V. CHAPTER S UMMARY The ma jor s ource of fructos e is s uc ro s e , which, whe n cle a ve d, re le a s e s e quimola r a mounts of fructos e a nd glucos e (Figure 12.8). Tra ns port of fructos e into ce lls is ins ulin inde pe nde nt. Fructos e is firs t phos phoryla te d to fruc to s e 1-pho s phate by fru c to kin a s e a nd the n cle a ve d by a ld o la s e B to dihydroxyac e tone phos phate a nd glyc e ralde hyde . The s e e nzyme s a re found in the live r, kidne y , a nd s mall inte s tinal muc o s a . A de fic ie nc y of fru c to kin a s e ca us e s a be nign condition (e s s e ntial fruc to s uria ), but a de fic ie nc y of a ldo la s e B ca us e s he re ditary fruc to s e into le ranc e (HFI), in which s e ve re hypo g lyc e mia a nd live r failure le a d to de ath if fructos e (a nd s ucros e ) in the die t is not e limina te d. Manno s e , a n importa nt compone nt of g lyc o pro te ins , is phos phoryla te d by h e xo kin a s e to manno s e 6-pho s phate , which is re ve rs ibly is ome rize d to fruc to s e 6-pho s phate by p h o s p h o m a n n o s e is o m e ra s e . Gluc o s e ca n be re duce d to s o rbito l (g luc ito l) by a ld o s e re d u c ta s e in ma ny tis s ue s , including the le ns , re tina , S c hwann c e lls , live r, kidne y , o varie s , a nd s e minal ve s ic le s . In ce lls of the live r, o varie s , a nd s e minal ve s ic le s , a s e cond e nzyme , s orbitol d e h yd ro g e n a s e , ca n oxidize s orbitol to produce fruc to s e . Hype rg lyc e mia re s ults in the a ccumula tion of s orbitol in thos e ce lls la cking s orbitol de hydroge na s e . The re s ulting o s mo tic e ve nts ca us e ce ll s we lling a nd ma y

V. Cha pte r S umma ry

143

contribute to the c atarac t fo rmatio n , pe riphe ral ne uropathy , ne phropathy , a nd re tinopathy s e e n in diabe te s . The ma jor die ta ry s ource of ga la ctos e is lac tos e . The tra ns port of ga la ctos e into ce lls is not ins ulin de pe nde nt. Ga la ctos e is firs t phos phoryla te d by g a la c to kin a s e (a de ficie ncy re s ults in ca ta ra cts ) to g alac to s e 1-pho s phate . This compound is conve rte d to uridine diphos phate (UDP)-g alac to s e by g a la c to s e 1-p h o s p h a te u rid yltra n s fe ra s e (GALT), with the nucle otide s upplie d by UDP-glucos e . A de fic ie nc y of this e nzyme ca us e s c las s ic g alac tos e mia . Ga la ctos e 1-phos pha te a ccumula te s , a nd e xce s s ga la ctos e is conve rte d to g alac tito l by a ldos e re duc ta s e . This ca us e s live r damag e , s e ve re inte lle c tual dis ability , a nd c atarac ts . Tre a tme nt re quire s re mova l of ga la ctos e (a nd la ctos e ) from the die t. For UDP-ga la ctos e to e nte r the ma ins tre a m of glucose me ta bolis m, it mus t firs t be conve rte d to UDP -glucos e by UDP-h e xo s e 4-e p im e ra s e . This e nzyme ca n a ls o be us e d to produce UDP -ga la ctos e from UDP-glucos e whe n the forme r is re quire d for the synthe s is of s tructura l ca rbohydra te s . Lac tos e is a dis a ccha ride tha t cons is ts of g alac tos e a nd gluc o s e . Milk a nd othe r da iry products a re the die ta ry s ource s of la ctos e . La ctos e is s ynthe s ize d by la c to s e s yntha s e from UDP-g alac to s e a nd g luc o s e in the lac tating mammary gland . The e nzyme ha s two s ubunits , pro te in A (which is a g a la c tos yltra n s fe ra s e found in mos t ce lls whe re it s ynthe s ize s N-ac e tyllac to s amine ) a nd prote in B (α-lac talbumin , which is found only in the la cta ting ma mma ry gla nds , a nd whos e s ynthe s is is s timula te d by the pe ptide hormone prolac tin ). Whe n both s ubunits a re pre s e nt, the tra ns fe ra s e produce s la ctos e .

Impo rtant die tary mo no s ac c haride s include

Fruc to s e

a re importa nt s ource s of

me ta bolize d by

S uc ro s e Fruits Hig h-fruc to s e c o rn s yrup

Milk and o the r lac to s e -c o ntaining dairy pro duc ts

a re importa nt s ource s of

me ta bolize d by

Glyc o lys is o r g luc o ne o g e ne s is

Glyc o lys is o r g luc o ne o g e ne s is Glycoge n

Galac to s e

providing

UDP-Gluc o s e

Ene rg y o r g luc o s e

providing

Galac to s e 1-P

Ene rg y o r g luc o s e

GALT Gluc o s e 1-P

by a pa thwa y us ing

Fruc to s e

Ald o la s e B

Fruc to s e 1-P

UDP-Galac to s e by a pa thwa y us ing

Gluc o ne o g e ne s is

Gluc o s e

GALT

Ald o la s e B Glyc e ralde hyde

is clinica lly importa nt be ca us e

Dihydro xyac e to ne P Mutatio ns in the g e ne fo r a ld o la s e B

is clinica lly importa nt be ca us e

Glyc e ralde hyde P Mutations in the g e ne fo r GALT

Glyc o lys is

le a d to

De fic ie nc y o f e nzyme ac tivity

Galac to s e

le a d to

Pyruvate le a ds to

He re ditary fruc to s e into le ranc e

Clas s ic g alac to s e mia

le a ds to

De fic ie nc y o f e nzyme ac tivity

Fig ure 12.8 Ke y conce pt ma p for me ta bolis m of fructos e a nd ga la ctos e . GALT = ga la ctos e 1-phos pha te uridylyltra ns fe ra s e ; UDP = uridine diphos pha te ; P = phos pha te .

144

12. Me ta bolis m of Monos a ccha ride s a nd Dis a ccha ride s

S tudy Que s tio ns Choos e the ONE be s t a ns we r. 12.1 A nurs ing fe ma le with cla s s ic ga la ctos e mia is on a ga la ctos e -fre e die t. S he is a ble to produce la ctos e in bre a s t milk be ca us e : A. ga la ctos e ca n be produce d from fructos e by is ome riza tion. B. ga la ctos e ca n be produce d from a glucos e me ta bolite by e pime riza tion. C. he xokina s e ca n e fficie ntly phos phoryla te ga la ctos e to ga la ctos e 1-phos pha te . D. the e nzyme a ffe cte d in ga la ctos e mia is a ctiva te d by a hormone produce d in the ma mma ry gla nd. 12.2 A 5-month-old boy is brought to his physicia n be ca us e o f vo m itin g , n ig h t s we a ts , a n d tre m o rs . His to ry re ve a le d tha t the se symptoms be ga n a fte r fruit juice s we re introduce d to his die t a s he wa s be ing we a ne d off bre a st milk. The physica l e xa mina tion wa s re ma rka ble for he pa tome ga ly. Te sts on the ba by’s urine were pos itive for re ducing s uga r but ne ga tive for glucos e . The infa nt most like ly suffe rs from a de ficie ncy of: A. B. C. D.

a ldola s e B. fructokina s e . ga la ctokina s e . β-ga la ctos ida s e .

12.3 La ctos e s ynthe s is is e s s e ntia l in the production of milk by ma mma ry gla nds . In la ctos e s ynthe s is : A. ga la ctos e from ga la ctos e 1-phos pha te is tra ns fe rre d to glucos e by ga la ctos yltra ns fe ra s e (prote in A), ge ne ra ting la ctos e . B. prote in A is us e d e xclus ive ly in the s ynthe s is of la ctos e . C. α -la cta lbumin (prote in B) re gula te s the s pe cificity of prote in A by incre a s ing its Km for glucos e . D. prote in B e xpre s s ion is s timula te d by prola ctin. 12.4 A 3-month-old girl is de ve loping ca ta ra cts . Othe r tha n not ha ving a s ocia l s mile or be ing a ble to tra ck obje cts vis ua lly, a ll othe r a s pe cts of the girl’s e xa mina tion a re norma l. Te s ts on the ba by’s urine a re pos itive for re ducing s uga r but ne ga tive for glucos e . Which e nzyme is mos t like ly de ficie nt in this girl? A. B. C. D.

Aldola s e B Fructokina s e Ga la ctokina s e Ga la ctos e 1-phos pha te uridylyltra ns fe ra s e

Corre ct a ns we r = B. Uridine diphos pha te (UDP )glucos e is conve rte d to UDP -ga la ctos e by UDP he xos e 4-e p im e r a s e , th e re b y p ro vidin g the a ppropria te form of ga la ctos e for la ctos e s ynthe s is . Is ome riza tion of fructos e to ga la ctos e doe s not occur in the huma n body. Ga la ctos e is not conve rte d to ga la ctos e 1-phos pha te by he xokina s e . A ga la ctos e -fre e die t provide s no ga la ctos e . Ga la ctos e mia is the re s ult of a n e nzyme de ficie ncy.

Corre ct a ns we r = A. The s ymptoms s ugge s t he re dita ry fructos e intole ra nce , a de ficie ncy in a ldola s e B. De ficie ncie s in fructokina s e or ga la ctokina s e re s ult in re la tive ly be nign cond itio n s c h a ra c te riz e d b y e le va te d le ve ls o f fructos e or ga la ctos e in the blood a nd urine . De ficie ncy in β-ga la ctos ida s e (la cta s e ) re s ults in a de cre a s e d a bility to de gra de la ctos e (milk s uga r). Conge nita l la cta s e de ficie ncy is quite ra re a nd would ha ve pre s e nte d much e a rlie r in this ba by (a nd with diffe re nt s ymptoms ). Typica l la cta s e de ficie ncy (a dult hypola cta s ia ) pre s e nts a t a la te r a ge .

C o rre c t a n s we r = D. Th e e xp re s s io n o f α -la cta lbumin (prote in B) is incre a s e d by the h o rm o n e p ro la c tin . Urid in e d ip h o s p h a te – ga la ctos e is the form us e d by the ga la ctos yltra n s fe ra s e (p ro te in A). P ro te in A is a ls o involve d in the s ynthe s is of the a mino s uga r, N-a c e tylla c to s a m in e . P ro te in B in c re a s e s the a ffinity of prote in A for glucos e a nd, s o, de cre a s e s the Km .

Corre ct a n s we r = C . Th e g irl is d e ficie n t in ga la ctokina s e a nd is una ble to a ppropria te ly phos phoryla te ga la ctos e . Ga la ctos e a ccumula te s in the blood (a nd urine ). In the le ns of the e ye , ga la ctos e is re duce d by a ldos e re ducta s e to ga la ctitol, a s uga r a lcohol, which ca us e s os motic e ffe cts tha t re s ult in ca ta ra ct forma tion. De ficie ncy of ga la ctos e 1-phos pha te uridylyltra ns fe ra s e a ls o re s ults in ca ta ra cts but is cha ra cte rize d by live r da ma ge a nd ne urologic e ffe cts . Fructokina s e de ficie ncy is a be nign condition. Aldola s e B de ficie ncy is s e ve re , with a ffe cts on s e ve ra l tis s ue s . Ca ta ra cts a re not typica lly s e e n.

Pe nto s e Pho s phate Pathway and Nic o tinamide Ade nine Dinuc le o tide Pho s phate

13

I. OVERVIEW The pe ntos e phos pha te pa thwa y (a ls o ca lle d the he xos e monophos pha te s hunt) occurs in the cytos ol of the ce ll. It include s two irre ve rs ible oxida tive re a ctions , followe d by a s e rie s of re ve rs ible s uga r–phos pha te inte rconve rs ions (Figure 13.1). No a de nos ine triphos pha te (ATP) is dire ctly cons ume d or produce d in the cycle . Ca rbon 1 of glucos e 6-phos pha te is re le a s e d a s CO 2 , a nd two re duce d nicotina mide a de nine dinucle otide phos pha te s (NADP Hs ) a re produce d for e a ch glucos e 6-phos pha te mole cule e nte ring the oxida tive pa rt of the pa thwa y. The ra te a nd dire ction of the re ve rs ible re a ctions of the pentos e phos pha te pa thwa y a re de te rmine d by the s upply of a nd de ma nd for inte rme dia te s of the cycle . The pa thwa y provide s a ma jor portion of the body’s NADP H, which functions a s a bioche mica l re ducta nt. It a ls o produce s ribos e 5-phos pha te , re quire d for the bios ynthe s is of nucle otide s (s e e p. 293), a nd provide s a me cha nis m for the me ta bolic us e of five -ca rbon s uga rs obta ine d from the die t or the de gra da tion of s tructura l ca rbohydra te s .

6-P Glucona te Ribulos e 5-P

A. De hydro g e natio n o f g luc o s e 6-pho s phate Glucos e 6-phos pha te de hydroge na s e (G6PD) ca ta lyze s a n irre ve rs ible oxida tion of glucos e 6-phos pha te to 6-phos phogluconola ctone in a re a ction tha t is s pe cific for oxidize d NADP (NADP +) a s the coe nzyme . The pe ntos e phos pha te pa thwa y is re gula te d prima rily a t the G6PD re a ction. NADP H is a pote nt compe titive inhibitor of the e nzyme , a nd, unde r mos t me ta bolic conditions , the ra tio of NADPH/

Ga la ctos e

UDP -Glucos e

6-P gluconola ctone

Ribos e 5-P

Ga la ctos e 1-P

Glucos e 1-P

Xylulos e 5-P

Glucos e 6-P

S e dohe ptulos e 7-P Erythros e 4-P

Fructos e 6-P

UDP -Ga la ctos e Glucos e Fructos e

Fructos e 1,6-bis -P Glyce ra lde hyde 3-P

Glyce ra lde hyde

Fructos e 1-P

Dihydroxya ce tone -P

Glyce ra lde hyde 3-P

Glyce rol-P

1,3-bis -P hos phoglyce ra te

Glyce rol

3-P hos phoglyce ra te Tria cylglyce rol 2-P hos phoglyce ra te

Ala Cys Gly Ser Thr Try

Fa tty a cyl CoA

Fa tty a cid

P hos phoe nolpyruva te La cta te CO 2

CO 2

NH3

P yruvate CO 2

Ma lonyl CoA

Ace tyl-CoA

Le u P he Tyr Trp Lys

Ace toa ce ta te

As n Ca rba moyl-P

β-Hydroxybutyra te As pa rta te

Citrulline

Ornithine

Oxa loa ce ta te

Citra te

Ma la te

Is ocitra te CO 2 α -Ke togluta ra te

Argininos uccina te Fuma ra te S uccina te

Arginine

Gln P ro His Arg

Glu

CO 2 S uccinyl CoA

Me thylma lonyl CoA

Ure a Ile Me t Va l Thr

P he Tyr

II. IRREVERS IBLE OXIDATIVE REACTIONS The oxida tive portion of the pe ntos e phos pha te pa thwa y cons is ts of thre e re a ctions tha t le a d to the forma tion of ribulos e 5-phos pha te , CO 2 , a nd two mole cule s of NADP H for e a ch mole cule of glucos e 6-phos pha te oxidize d (Figure 13.2). This portion of the pa thwa y is pa rticula rly importa nt in the live r, la cta ting ma mma ry gla nds , a nd a dipos e tis s ue , which a re a ctive in the NADP H-de pe nde nt bios ynthe s is of fa tty a cids (s e e p. 186); in the te s te s , ova rie s , pla ce nta , a nd a dre na l corte x, which a re a ctive in the NADP H-de pe nde nt bios ynthe s is of s te roid hormone s (s e e p. 237); a nd in re d blood ce lls (RBCs ), which re quire NADP H to ke e p gluta thione re duce d (s e e p. 152).

Glycoge n

P ropionyl CoA Ace tyl CoA Fa tty a cyl CoA (odd-numbe r ca rbons )

6-P Glucona te Ribulos e 5-P

6-P Gluconola ctone

Ribos e 5-P

Xylulos e 5-P

Glucos e 6-P

S e dohe ptulos e 7-P Erythros e 4-P

Fructos e 6-P Fructos e 1,6-bis -P DHAP

Glyce ra lde hyde 3-P

Glyce ra lde hyde 3-P

Fig ure 13.1 P e ntos e phos pha te pa thwa y s hown a s a compone nt of the me ta bolic ma p (s e e Figure 8.2, p. 92 for a more de ta ile d vie w of the me ta bolic pa thwa ys ). P = phos pha te ; DHAP = dihydroxya ce tone phos pha te .

145

146

13. P e ntos e P hos pha te P a thwa y a nd Nicotina mide Ade nine Dinucle otide P hos pha te NADP + is s ufficie ntly high to s ubs ta ntia lly inhibit e nzyme a ctivity. Howe ve r, with incre a s e d de ma nd for NADP H, the ra tio of NADP H/ NADP + de cre a s e s , a nd flux through the cycle incre a s e s in re spons e to the enha nce d a ctivity of G6PD. Ins ulin upre gula te s e xpre s s ion of the ge ne for G6P D, a nd flux through the pa thwa y incre a s e s in the a bs orptive s ta te (s e e p. 323). B. Fo rmatio n o f ribulo s e 5-pho s phate 6-P hos phogluconola ctone is hydrolyze d by 6-phos phogluconola ctone hydrola s e . The re a ction is irre ve rs ible a nd not ra te limiting. The oxida tive de ca rboxyla tion of the product, 6-phos phoglucona te , is ca ta lyze d by 6-phos phoglucona te de hydroge na s e . This irre ve rs ible re a ction produce s a pe ntos e s uga r–phos pha te (ribulos e 5-phos pha te ), CO 2 (from ca rbon 1 of glucos e ), a nd a s e cond mole cule of NADP H (s e e Figure 13.2).

Tra ns ke tola s e

Tra ns ke tola s e H

H

Re duc tive anabo lic pathways

∆2C

C H H C OH

Nuc le ic ac id bio s ynthe s is

NADP H, H+

NADP H, H+

O HO

C C C

Tra ns a ldola s e

OH

∆2C

∆3C

O H

H

O

H C OH

H C OH

H C OH

C H H C OH

H C OH

H C OH

H C OH

H C O

H C O

P

H

H C O

P

C C

OH O

HO C H H C OH H C O

P

P

H

H

H

H

Ribo s e 5pho s phate

S e do he ptulo s e 7-pho s phate

Erythro s e 4pho s phate

Xylulo s e 5pho s phate

4 O

NADP +

HO C H

P

H Gluc o s e 6pho s phate

CO 2

HO C H

1,2

H C OH

H

H C OH

H2 O

H C OH

H C O

NADP +

C O-

C H H C OH

O

H C OH

3

H C OH H C O

P

H

6-Pho s pho g luc o nate

H C OH

7

H

H C OH

C O H C OH

C O HO C H 5

H C OH H C O

6

H

H C OH H C O

P

H

H

Ribulo s e 5pho s phate

Xylulo s e 5pho s phate

Oxidative re ac tio ns (irre ve rs ible )

O

P

HO

C C C

8

H

H OH

H

O

C C

OH O

HO C H

H

O

C H

H C OH

H C OH

C H

H C OH

H C OH

H C OH

H C OH

H C O

P

H C O

P

H

H

Glyc e ralde hyde 3-pho s phate

Fruc to s e 6pho s phate

H C O

P

H C O

H

P

H

Fruc to s e 6- Glyc e rpho s phate alde hyde 3-pho s phate

No no xidative re ac tio ns (re ve rs ible ) Glyc o lytic pathway ∆2C

Fig ure 13.2 Re a ctions of the pe ntos e phos pha te pa thwa y. Enzyme s numbe re d a bove a re : 1, 2) glucos e 6-phos pha te de hydroge na s e a nd 6-phos phogluconola ctone hydrola s e , 3) 6-phos phoglucona te de hydroge na s e , 4) ribos e 5-phos pha te is ome ra s e , 5) phos phope ntos e e pime ra s e , 6 a nd 8) tra ns ke tola s e (coe nzyme : thia mine pyrophos pha te ), a nd 7) tra ns a ldola s e . ∆2C = two ca rbons a re tra ns fe rre d in tra ns ke tola s e re a ctions ; ∆3C = thre e ca rbons a re tra ns fe rre d in the tra ns a ldola s e re a ction. This ca n be re pre s e nte d a s : 5C s uga r + 5C s uga r ∆2C 7C s uga r + 3C s uga r ∆3C 4C s uga r + 6C s uga r. NADP (H) = nicotina mide a de nine dinucle otide phos pha te ; P = phos pha te .

IV. Us e s of NADP H

147

III. REVERS IBLE NONOXIDATIVE REACTIONS 6-P Glucona te

The nonoxida tive re a ctions of the pe ntos e phos pha te pa thwa y occur in a ll ce ll type s s ynthe s izing nucle otide s a nd nucle ic a cids . The s e re a ctions ca ta lyze the inte rconve rs ion of s uga rs conta ining thre e to s e ve n ca rbons (s e e Figure 13.2). The s e re ve rs ible re a ctions pe rmit ribulos e 5-phos pha te (produce d by the oxida tive portion of the pa thwa y) to be conve rte d e ithe r to ribos e 5-phos pha te (ne e de d for nucle otide s ynthe s is ; s e e p. 293) or to inte rme dia te s of glycolys is (tha t is , fructos e 6-phos pha te a nd glyce ra lde hyde 3-phos pha te ). For e xa mple , ma ny ce lls tha t ca rry out re ductive bios ynthe tic re a ctions ha ve a gre a te r ne e d for NADP H tha n for ribos e 5-phos pha te . In this ca s e , tra ns ke tola s e (which tra ns fe rs two-ca rbon units in a thia mine pyrophos pha te [TP P ]re quiring re a ction)a nd tra ns a ldola s e (which tra ns fe rs thre e -ca rbon units ) conve rt the ribulos e 5-phos pha te produce d a s a n e nd product of the oxida tive re a ctions to glyce ra lde hyde 3-phos pha te a nd fructos e 6-phos pha te , which a re glycolytic inte rme dia te s . In contra s t, whe n the de ma nd for ribos e for nucle otide s a nd nucle ic a cids is gre a te r tha n the ne e d for NADP H, the nonoxida tive re a ctions ca n provide the ribos e 5-phos pha te from glyce ra lde hyde 3-phos pha te a nd fructos e 6-phos pha te in the a bs e nce of the oxida tive s te ps (Figure 13.3).

6-P Gluconola ctone

Ribulo s e 5-P

Glucos e

Ribo s e 5-P

Glucos e 6-P

Xylulo s e 5-P

Fruc to s e 6-P Fructos e 1,6-bis -P

S e do he ptulo s e 7-P

Erythro s e 4-P

DHAP Glyc e ralde hyde 3-P Glyc e ralde hyde 3-P GLYCOLYS IS

Fig ure 13.3 Forma tion of ribos e 5-phos pha te from inte rme dia te s of glycolys is . P = phos pha te ; DHAP = dihydroxya ce tone phos pha te .

In a ddition to tra ns ke tola s e s , TP P is re quire d by the e nzyme comple xe s pyruva te de hydroge na s e (s e e p. 110), α -ke togluta ra te de hydroge na s e of the citric a cid cycle (s e e p. 112), a nd bra nche d-cha in α -ke to a cid de hydroge na s e of bra nche d-cha in a mino a cid ca ta bolis m (s e e p. 266).

IV. US ES OF NADPH The coe nzyme NADP H diffe rs from nicotina mide a de nine dinucle otide (NADH) only by the pre s e nce of a phos pha te group on one of the ribos e units (Figure 13.4). This s e e mingly s ma ll cha nge in s tructure a llows NADPH to inte ra ct with NADPH-s pe cific e nzyme s tha t ha ve unique role s in the ce ll. For e xa mple , in the cytos ol of he pa tocyte s the s te a dy-s ta te ra tio of NADP +/NADP H is a pproxima te ly 0.1, which fa vors the us e of NADPH in re ductive bios ynthe tic re a ctions . This contra s ts with the high ra tio of NAD+/NADH (a pproxima te ly 1000), which fa vors a n oxida tive role for NAD+. This s e ction s umma rize s s ome importa nt NADP + a nd NADPHs pe cific functions in re ductive bios ynthe s is a nd detoxifica tion re a ctions .

H H

O NH2

O

N

O

O P OO

HO

OH N

O P OO

O

N

NH2 N N

A. Re duc tive bio s ynthe s is NADP H ca n be thought of a s a high-e ne rgy mole cule , much in the s a me wa y a s NADH. Howe ve r, the e le ctrons of NADP H a re de s tine d for us e in re ductive bios ynthe s is , ra the r tha n for tra ns fe r to oxyge n a s is the ca s e with NADH (s e e p. 74). Thus , in the me ta bolic tra ns forma tions of the pe ntos e phos pha te pa thwa y, pa rt of the e ne rgy of glucos e 6-phos pha te is cons e rve d in NADP H, a mole cule with a ne ga tive re duction pote ntia l (s e e p. 77), tha t, the refore , ca n be us e d in re a ctions re quiring a n e le ctron donor, s uch a s fa tty a cid (s e e p. 186) a nd s te roid (s e e p. 237) s ynthe s is .

HO

OP O3 2 –

Fig ure 13.4 S tructure of re duce d nicotina mide a de nine dinucle otide phos pha te (NADP H).

148

13. P e ntos e P hos pha te P a thwa y a nd Nicotina mide Ade nine Dinucle otide P hos pha te

e-

A

e-

-

O2 Oxyg e n

O2 S upe ro xide

H2 O2 Hydro g e n pe ro xide

B

e-

eOH Hydro xyl radic al

H2 O Wate r

Ca ta la s e

-

H2 O2 Hydro g e n pe ro xide

O2 S upe ro xide

O2 Oxyg e n

S u p e ro xid e d is m u ta s e

OH Hydro xyl radic al

H2 O Wate r

Glu ta th io n e p e ro xid a s e

2 G-S H

G-S -S -G

Fig ure 13.5 A. Forma tion of re a ctive inte rme dia te s from mole cular oxyge n. e – = e le ctrons . B. Actions of a ntioxida nt e nzyme s. G-SH = re duce d gluta thione ; G-S-S -G = oxidize d gluta thione . (S ee Figure 13.6B for the re ge ne ra tion of G-SH.)

B. Re duc tio n o f hydro g e n pe ro xide

A

COO -

HS G-S H

Glyc ine

CH2 HN C O CH2 CH HN

Cys te ine

C O CH2 CH2 HCNH3 COO -

+

Glutamate

B NADPH + H+

G-S –S -G (oxidize d)

Glu ta th io n e p e ro xid a s e

Glu ta th io n e re d u c ta s e

NADP +

2 H2 O

2 G-S H (re duce d)

H2 O2

Fig ure 13.6 A. S tructure of re duce d gluta thione (G-S H). [Note : Gluta ma te is linke d to cys te ine through a γ-ca rboxyl, ra the r tha n a n α -ca rboxyl.] B. Gluta thione -me dia te d re duction of hydroge n pe roxide (H2 O 2 ) by re duce d nicotina mide a de nine dinucle otide phos pha te (NADP H). G-S -S -G = oxidize d gluta thione .

Hydroge n pe roxide (H2 O 2 ) is one of a fa mily of re a ctive oxyge n s pe cie s (ROS ) tha t a re forme d from the pa rtia l re duction of mole cula r oxyge n (Figure 13.5A). The s e compounds a re forme d continuous ly a s byproducts of a e robic me ta bolis m, through re a ctions with drugs a nd e nvironme nta l toxins , or whe n the le ve l of a ntioxida nts is diminis he d, a ll cre a ting the condition of oxida tive stre s s . The highly re a ctive oxyge n inte rme dia te s ca n ca us e s e rious che mica l da ma ge to DNA, prote ins , a nd uns a tura te d lipids a nd ca n le a d to ce ll de a th. ROS ha ve be e n implica te d in a numbe r of pa thologic proce s s e s , including re pe rfus ion injury, ca nce r, infla mma tory dis e a s e , a nd a ging. The ce ll ha s s e ve ra l prote ctive me cha nis ms tha t minimize the toxic pote ntia l of the s e compounds . 1. Enzyme s that c atalyze antio xidant re ac tio ns : Re duce d gluta thi-

one (G-S H), a tripe ptide -thiol (γ-gluta mylcys te inylglycine ) pre s e nt in mos t ce lls , ca n che mica lly de toxify H2 O 2 (Figure 13.5B). This re a ction, ca ta lyze d by the s e le nium-conta ining gluta thione pe roxida s e , forms oxidize d gluta thione (G-S -S -G), which no longe r ha s prote ctive prope rtie s . The ce ll re ge ne ra te s G-S H in a re action ca ta lyze d by gluta thione re ducta s e , us ing NADP H a s a s ource of re ducing e quiva le nts . Thus , NADP H indire ctly provide s e le ctrons for the re duction of H2 O 2 (Figure 13.6). [Note : RBCs a re tota lly de pe nde nt on the pe ntos e phos pha te pa thwa y for the ir supply of NADP H be ca us e , unlike othe r ce ll type s , RBCs do not ha ve a n a lte rna te s ource for this e s s e ntia l coe nzyme .] Additiona l e nzyme s , s uch a s s upe roxide dis muta s e a nd ca ta la s e , ca ta lyze the conve rs ion of othe r re a ctive oxyge n inte rme dia te s to ha rmle s s products (s e e Figure 13.5B). As a group, the s e e nzyme s s e rve a s a de fe ns e s ys te m to gua rd a ga ins t the toxic e ffe cts of ROS . 2. An tio x id a n t c h e m ic a ls : A n u m b e r o f in tra c e llu la r re d u c -

ing a ge nts , s uch a s a s corba te (s e e p. 377), vita min E (s e e p. 391), a nd β-ca rote ne (s e e p. 382), a re a ble to re duce a nd, the re by, de toxify re a ctive oxyge n inte rme dia te s in the la bora tory. Cons umption of foods rich in the s e a ntioxida nt compounds ha s be e n corre la te d with a re duce d ris k for ce rta in type s of ca nce rs a s we ll a s de cre a s e d fre que ncy of ce rta in othe r chronic he a lth

IV. Us e s of NADP H proble ms . The re fore , it is te mpting to s pe cula te tha t the e ffe cts of the s e compounds a re , in pa rt, a n e xpre s s ion of the ir a bility to que nch the toxic e ffe ct of ROS . Howe ve r, clinica l tria ls with a ntioxida nts a s die ta ry s upple me nts ha ve fa ile d to s how cle a r be ne ficia l e ffe cts . In the ca s e of die ta ry s upple me nta tion with β-ca rote ne , the ra te of lung ca nce r in s moke rs incre a s e d ra the r tha n de cre a s e d. Thus , the he a lth-promoting e ffe cts of die ta ry fruits a nd ve ge ta ble s like ly re fle ct a comple x inte ra ction a mong ma ny na tura lly occurring compounds , which ha s not be e n duplica te d by cons umption of is ola te d a ntioxida nt compounds .

149

NADPH + H+

e– Cyto c h ro m e P 450 re d u c ta s e FAD, FMN S ubs trate A-H P450-Fe 3+ A-H

C. Cyto c hro me P450 mo no o xyg e nas e s ys te m Monooxyge na s e s (mixe d-function oxida s e s ) incorpora te one a tom from mole cula r oxyge n into a s ubs tra te (cre a ting a hydroxyl group), with the othe r a tom be ing re duce d to wa te r. In the cytochrome P 450 monooxyge na s e s ys te m, NADP H provide s the re ducing e quiva le nts re quire d by this s e rie s of re a ctions (Figure 13.7). This s ys te m pe rforms diffe re nt functions in two s e pa ra te loca tions in ce lls . The ove ra ll re a ction ca ta lyze d by a cytochrome P 450 e nzyme is : R-H + O 2 + NADP H + H+ → R-OH + H2 O + NADP + whe re R ma y be a s te roid, drug, or othe r che mica l. [Note : Cytochrome P 450 (CYP ) e nzyme s a re a ctua lly a s upe rfa mily of re la te d, he me -conta ining monooxyge na s e s tha t pa rticipa te in a broa d va rie ty of re a ctions . The P450 in the na me re fle cts the a bs orba nce a t 450 nm by the prote in.]

NADP +

e–

P450-Fe 3+

P450-Fe 2+ A-H

H2 O

O2

2H+ P450-Fe 3+ A-H O2–

P450-Fe 3+ O2=

A-O H Pro duc t

e– Cyto c h ro m e P 450 re d u c ta s e FAD, FMN

e–

1. Mito c ho ndrial s ys te m: An importa nt function of the cytochrome

P 450 monooxyge na s e s ys te m found a s s ocia te d with the inne r mitochondria l me mbra ne is the bios ynthe s is of s te roid hormone s . In s te roidoge nic tis s ue s , s uch a s the pla ce nta , ova rie s , te s te s , a nd a dre na l corte x, it is us e d to hydroxyla te inte rme dia te s in the conve rs ion of chole s te rol to s te roid hormone s , a proce s s tha t ma ke s the s e hydrophobic compounds more wa te r s oluble (s e e p. 237). The live r us e s this s a me s ys te m in bile a cid s ynthes is (s e e p. 224) a nd the hydroxyla tion of chole ca lcife rol to 25-hydroxychole ca lcife rol (vita min D3 ; s e e p. 386), a nd the kidne y us e s it to hydroxyla te vita min D3 to its biologica lly a ctive 1,25-dihydroxyla te d form. 2. Mic ro s o mal s ys te m: An e xtre me ly importa nt function of the micro-

s oma l cytochrome P 450 monooxyge na s e s ys te m found a s s ocia te d with the me mbra ne of the s mooth e ndopla s mic re ticulum (pa rticula rly in the live r) is the de toxifica tion of fore ign compounds (xe nobiotics ). The s e include nume rous drugs a nd s uch va rie d polluta nts a s pe trole um products a nd pe s ticide s . CYP e nzyme s of the micros oma l s ys te m (for e xa mple , CYP 3A4), ca n be us e d to hydroxyla te the s e toxins . The purpos e of the s e modifica tions is two-fold. Firs t, it ma y its e lf a ctiva te or ina ctiva te a drug a nd s e cond, ma ke a toxic compound more s oluble , the re by fa cilita ting its e xcre tion in the urine or fe ce s . Fre que ntly, howe ve r, the ne w hydroxyl group will s e rve a s a s ite for conjuga tion with a pola r mole cule , s uch a s glucuronic a cid (s e e p. 161), which will s ignifica ntly incre a s e the compound’s s olubility. [Note : Polymorphis ms (s e e p.473) in the ge ne s for CYP e nzyme s ca n le a d to diffe re nce s in drug me ta bolis m.]

NADP +

NADPH + H+

Fig ure 13.7 Cytochrome P 450 monooxyge na s e cycle (s implifie d). Ele ctrons (e −) move from NADP H to FAD to FMN of the re ducta s e a nd the n to the he me iron (Fe ) of the P 450 e nzyme . [Note : In the mitochondria l s ys te m, e le ctrons move from FAD to a n irons ulfur prote in a nd the n to the P 450 e nzyme .] FAD = fla vin a de nine dinucle otide ; FMN = fla vin mononucle otide ; NADP H = re duce d nicotina mide a de nine dinucle otide phos pha te .

150

13. P e ntos e P hos pha te P a thwa y a nd Nicotina mide Ade nine Dinucle otide P hos pha te

Attac hme nt o f the patho g e n to a phag o c ytic c e ll

1

BACTERIUM

Ig G

Ing e s tio n o f the mic ro o rg anis m

2

D. Phag o c yto s is by white blo o d c e lls P ha gocytos is is the inge s tion by re ce ptor-me dia te d e ndocytos is of microorga nis ms , fore ign pa rticle s , a nd ce llula r de bris by ce lls s uch a s ne utrophils a nd ma cropha ge s (monocyte s ). It is a n importa nt de fe ns e me cha nis m, pa rticula rly in ba cte ria l infe ctions . Ne utrophils a nd monocyte s a re a rme d with both oxyge n-inde pe nde nt a nd oxyge n-de pe nde nt me cha nis ms for killing ba cte ria . 1. Oxyg e n-inde pe nde nt me c hanis m: Oxyge n-inde pe nde nt me ch-

Ig G re c e pto r

a nis ms us e pH cha nge s in pha golys os ome s a nd lys os oma l e nzyme s to de s troy pa thoge ns . 2. Oxyg e n-de pe nde nt s ys te m: Oxyge n-de pe nde nt me cha nis ms

Lys o s o me Vac uo le fo rmatio n

Phag o s o me Phag o lys o s o me

De s truc tio n o f the mic ro o rg anis m

3

O2 NADPH RES PIRATORY BURS T

NADP H oxida s e NADP +

O2 – S ponta ne ous ly or by s upe roxide dis muta s e H2 O2 Mye lope roxida s e HOCl OH•

Fe 2 + Fe 3 +

B ACT ER IU M

B ACTE R

IUM

Cl–

Fig ure 13.8 P ha gocytos is a nd the oxyge nde pe nde nt pa thwa y of microbia l killing. IgG = the a ntibody immunoglobulin G; NADP H = re duce d nicotina mide a de nine dinucle otide phos pha te ; O 2 – = s upe roxide ; HOCl = hypochlorous a cid; OH• = hydroxyl ra dica l.

include the e nzyme s NADPH oxida s e a nd mye lope roxida s e (MPO) tha t work toge the r in killing ba cte ria (Figure 13.8). Ove ra ll, the MP O s ys te m is the mos t pote nt of the ba cte ricida l me chanis ms . An inva ding ba cte rium is re cognize d by the immune sys te m a nd a tta cke d by a ntibodie s tha t bind it to a re ce ptor on a pha gocytic ce ll. Afte r inte rna liza tion of the microorga nis m ha s occurre d, NADP H oxida s e , loca te d in the le ukocyte ce ll me mbra ne , is a ctiva te d a nd re duce s O 2 from the s urrounding tis s ue to s upe roxide (O 2 –• ), a fre e ra dica l, a s NADP H is oxidize d. The ra pid cons umption of O 2 tha t a ccompa nie s forma tion of O 2 –• is re fe rre d to a s the “re s pira tory burs t.” [Note : Active NADP H oxida s e is a me mbra ne a s s ocia te d comple x conta ining a fla vocytochrome plus a dditiona l pe ptide s tha t tra ns loca te from the cytopla s m upon a ctiva tion of the le ukocyte . Ele ctrons move from NADP H to O 2 via fla vin a de nine nucle otide (FAD) a nd he me , ge ne ra ting O 2 –•. Ra re ge ne tic de ficie ncie s in NADPH oxida s e ca us e chronic granuloma tous dis e a s e (CGD) cha ra cte rize d by s e ve re , pe rs is te nt infe ctions a nd the forma tion of gra nuloma s (nodula r a re a s of infla mma tion) tha t s e que s te r the ba cte ria tha t we re not de s troye d.] Ne xt, O 2 –• is conve rte d to H2 O 2 (a ROS ), e ithe r s ponta ne ous ly or ca ta lyze d by s upe roxide dis muta s e . In the pre s e nce of MPO, a he me -conta ining lys os oma l e nzyme pre s e nt within the pha golys os ome , pe roxide plus chloride ions are conve rte d to hypochlorous a cid ([HOCl] the ma jor compone nt of hous e hold ble a ch), which kills the ba cte ria . The pe roxide ca n als o be pa rtia lly re duce d to the hydroxyl ra dica l (OH•), a ROS, or be fully re duce d to wa te r by ca ta la s e or gluta thione pe roxida s e. [Note: De ficie ncie s in MP O do not confe r incre a s e d s us ce ptibility to infe ction be ca us e pe roxide from NADPH oxida s e is ba cte ricida l.] E. S ynthe s is o f nitric o xide Nitric oxide (NO) is re cognize d a s a me dia tor in a broa d a rra y of biologic s yste ms . NO is the e ndothe lium-de rive d re la xing fa ctor, which ca us e s va s odila tion by re la xing va s cula r s mooth mus cle . NO a ls o a cts a s a ne urotra ns mitte r, pre ve nts pla te le t a ggrega tion, a nd pla ys a n e s s e ntia l role in ma cropha ge function. NO ha s a ve ry s hort ha lf-life in tis s ue s (3–10 s e conds ) be ca us e it re a cts with oxyge n a nd s upe roxide a nd the n is conve rte d into nitra te s a nd nitrite s including pe roxynitrite (O=NOO – ), a re a ctive nitroge n s pe cie s (RNS ). [Note : NO is

IV. Us e s of NADP H a fre e ra dica l ga s tha t is ofte n confus e d with nitrous oxide (N2 O), the “la ughing ga s ” tha t is us e d a s a n a ne s the tic a nd is che mica lly sta ble .] 1. Nitric oxide s ynthas e: Arginine, O 2, and NADPH are substrates for

cytosolic NO synthase ([NOS] Figure 13.9). Flavin mononucleotide (FMN), FAD, heme, and tetrahydrobiopterin (see p. 268) are coenzyme s , a nd NO a nd citrulline a re products of the rea ction. Thre e NOS, each the product of a different gene, have been identified. Two are constitutive (synthesized at a constant rate), Ca 2+–calmodulindependent enzymes (see p. 133). They are found primarily in endothelium (eNOS) and neural tissue (nNOS) and constantly produce ve ry low le ve ls of NO for va s odila tion a nd ne urotra nsmis s ion. An inducible , Ca 2+-inde pe nde nt e nzyme (iNOS) ca n be e xpre ss e d in ma ny ce lls , including ma cropha ge s a nd ne utrophils , a s a n e a rly de fe ns e a ga ins t pa thoge ns . The s pe cific induce rs for iNOS va ry with cell type, and include proinflammatory cytokines, such as tumor ne cros is fa ctor-α (TNF-α) a nd inte rfe ron-γ (IFN-γ), a nd ba cte ria l e ndotoxins s uch a s lipopolys a ccha ride (LP S ). The s e compounds promote synthesis of iNOS, which can result in large amounts of NO being produced over hours or even days. 2. Ac tio ns o f nitric o xide o n vas c ular e ndo the lium: NO is a n impor-

ta nt me dia tor in the control of va s cula r s mooth mus cle tone . NO is s ynthe s ize d by e NOS in e ndothe lia l ce lls a nd diffus e s to va s cula r s mooth mus cle , whe re it a ctiva te s the cytos olic form of gua nyla te cycla s e (a ls o known a s gua nylyl cycla s e ) to form cyclic gua nos ine monophos pha te (cGMP ). [Note : This re a ction is a na logous to the forma tion of cyclic AMP by a de nyla te cycla s e (s e e p. 94), e xce pt tha t this gua nyla te cycla s e is not me mbra ne a s s ocia te d.] The re s ulta nt ris e in cGMP ca us e s a ctiva tion of prote in kina s e G, which phos phoryla te s Ca 2+ cha nne ls , ca us ing de cre a s e d e ntry of Ca 2+ into s mooth mus cle ce lls . This de cre a s e s the ca lcium–ca lmodulin a ctiva tion of myos in light-cha in kinas e , the re by de cre a s ing s mooth mus cle contra ction a nd fa voring re la xa tion. Va s odila tor nitra te s , s uch a s nitroglyce rin, a re meta bolize d to NO, which ca us e s re la xa tion of va s cula r s mooth mus cle a nd, the re fore , lowe rs blood pre s s ure . Thus , NO ca n be e nvis ione d a s a n e ndoge nous nitrova s odila tor. [Note : NO is involve d in pe nile e re ction. S ilde na fil citra te , us e d in the tre a tme nt of ere ctile dys function, inhibits the phos phodie s te ra s e tha t ina ctiva te s cGMP .]

151

+ NADPH NADP NH2 NH2 + + H C N H2 + C O NH NH CH2 CH2 NO s yn th a s e (NOS ) CH2 CH2 CH2 CH2 HCNH3 + HCNH3 + O2 COO COO L-Arg inine L-Citrulline

NO

Nitric o xide

Re laxe s s mo o th mus c le

Pre ve nts plate le t ag g re g atio n

Func tio ns as a ne uro trans mitte r in brain

3. Ro le o f nitric o xide in mac ro phag e bac te ric idal ac tivity: In

ma cropha ge s , iNOS a ctivity is norma lly low, but s ynthe s is of the e nzyme is s ignifica ntly s timula te d by ba cte ria l LP S a nd by re le a s e of IFN-γ a nd TNF-α in re s pons e to the infe ction. Activa te d ma cropha ge s form O 2 –• ra dica ls (s e e p. 150) tha t combine with NO to form inte rme dia te s tha t de compos e , producing the highly ba cte ricida l OH• ra dica l. 4. Othe r func tio ns o f nitric o xide : NO is a pote nt inhibitor of pla te -

le t a dhe s ion a nd a ggre ga tion (by a ctiva ting the cGMP pa thwa y). It is a ls o cha ra cte rize d a s a ne urotra ns mitte r in the ce ntra l a nd pe riphe ra l ne rvous s ys te ms .

Me diate s tumo ric idal and bac te ric idal ac tio ns o f mac ro phag e s

Fig ure 13.9 S ynthe s is a nd s ome of the a ctions of nitric oxide (NO). NADP H = re duce d nicotina mide a de nine dinucle otide phos pha te . [Note : Fla vin mononucle otide , fla vin a de nine dinucle otide , he me , a nd te tra hydrobiopte rin a re a dditiona l coe nzyme s re quire d by NOS .]

152

13. P e ntos e P hos pha te P a thwa y a nd Nicotina mide Ade nine Dinucle otide P hos pha te

ERYTHROCYTE E

Glu c o s e 6-p h o s p h a te d e h yd ro g e n a s e de fic ie nc y impairs the ability o f an e rythro c yte to fo rm NADPH, re s ulting in he mo lys is .

Gluc uc o s e + NADP

Glucc o s e 6-pho s phate

O Oxidant s tre s s C Ce rtain drug s IInfe c tio ns F Fava be ans

2 G-S H

H2 O2

2 ADP Glu c o s e 6-p h o s p h a te d e h yd ro g e n a s e

Glyc o lytic pathway

PPP

2 ATP

6-Pho s pho g luc o no lac to ne

Glu ta th io n e re d u c ta s e

NADPH + H+

Glu ta th io n e p e ro xid a s e G-S -S -G

2 H2 O

2 Lac tate

Fig ure 13.10 P a thwa ys of glucos e 6-phos pha te me ta bolis m in the erythrocyte . NADP(H) = nicotina mide a de nine dinucle otide phos pha te ; G-SH = re duce d gluta thionine ; G-S-S-G = oxidize d gluta thionine ; PP P = pe ntos e phos pha te pa thwa y.

V. GLUCOS E 6-PHOS PHATE DEHYDROGENAS E DEFICIENCY G6P D de ficie ncy is a he re dita ry dis e a s e cha ra cte rize d by he molytic a ne mia ca us e d by the ina bility to de toxify oxidizing a ge nts . G6P D de ficie ncy is the mos t common dis e a s e -producing e nzyme a bnorma lity in huma ns , a ffe cting more tha n 400 million individua ls worldwide . This de ficie ncy ha s the highe s t pre va le nce in the Middle Ea s t, tropica l Africa a nd As ia , a nd pa rts of the Me dite rra ne a n. G6P D de ficie ncy is X linke d a nd is , in fa ct, a fa mily of de ficie ncie s ca us e d by a numbe r of diffe re nt muta tions in the ge ne coding for G6P D. Only s ome of the re s ulting prote in va ria nts ca us e clinica l s ymptoms . [Note : In a ddition to he molytic a ne mia , a clinica l ma nife s ta tion of G6P D de ficie ncy is ne ona ta l ja undice a ppe a ring 1–4 da ys a fte r birth. The ja undice , which ma y be s e ve re , typica lly re s ults from incre a s e d production of unconjuga te d bilirubin (s e e p. 285).] The life s pa n of individua ls with a s e ve re form of G6P D de ficie ncy ma y be s ome wha t s horte ne d a s a re s ult of complica tions a ris ing from chronic he molys is . This ne ga tive e ffe ct of G6P D de ficie ncy ha s be e n ba la nce d in e volution by a n a dva nta ge in s urviva l—a n incre a s e d re s is ta nce to P la s modium fa lcipa rum ma la ria . [Note : S ickle ce ll tra it a nd β-tha la s s e mia minor a ls o confe r re s is ta nce to ma la ria .] A. Ro le o f g luc o s e 6-pho s phate de hydro g e nas e in re d blo o d c e lls He inz bo die s

Fig ure 13.11 He inz bodie s in e rythrocyte s of a pa tie nt with glucos e 6-phos pha te de hydroge na s e de ficie ncy.

Diminis he d G6P D a ctivity impa irs the a bility of the ce ll to form the NADPH that is essential for the maintena nce of the G-SH pool. This results in a de crea se in the cellular detoxifica tion of fre e radica ls and pe roxide s forme d within the ce ll (Figure 13.10). G-S H a ls o he lps maintain the reduced sta tes of sulfhydryl groups in prote ins, including hemoglobin. Oxida tion of those sulfhydryl groups le ads to the forma tion of de na ture d prote ins tha t form ins oluble ma s se s (ca lle d He inz bodie s ) tha t a tta ch to RBC me mbra ne s (Figure 13.11). Additiona l oxida tion of membra ne proteins causes RBCs to be rigid (less deformable ), and they are removed from the circulation by macrophage s in the spleen a nd liver. Although G6PD deficiency occurs in all ce lls of the affected individual, it is most se vere in RBCs, where the pentose phos pha te pathway provide s the only me a ns of ge nera ting NADPH.

V. Glucos e 6-P hos pha te De hydroge na s e De ficie ncy Other tissues have alternative source s for NADPH production (such as NADP +-dependent mala te dehydrogenase [malic enzyme]; see p. 186) that can keep G-SH reduced. The RBC ha s no nucleus or ribosomes a nd ca nnot renew its supply of the enzyme. Thus, RBCs are particularly vulne rable to enzyme variants with diminishe d stability. B. Pre c ipitating fac to rs in g luc o s e 6-pho s phate de hydro g e nas e de fic ie nc y Mos t individua ls who ha ve inhe rite d one of the G6PD muta tions do not s how clinica l ma nife s ta tions (tha t is , the y a re a s ymptoma tic). Howe ve r, s ome pa tie nts with G6P D de ficie ncy de ve lop he molytic a ne mia if the y a re tre a te d with a n oxida nt drug, inge s t fa va be a ns , or contra ct a s e ve re infe ction. 1. Oxidant drug s : Commonly us e d drugs tha t produce he molytic

a ne mia in pa tie nts with G6P D de ficie ncy a re be s t re me mbe re d from the mne monic AAA: a ntibiotics (for e xa mple , s ulfa me thoxa zole a nd chlora mphe nicol), a ntima la ria ls (for e xa mple , prima quine but not chloroquine or quinine ), a nd a ntipyre tics (for e xa mple , a ce ta nilid but not a ce ta minophe n).

153

Clas s

Re s idual e nzyme ac tivity

Clinic al s ympto ms

I

Ve ry s e ve re (c hro nic he mo lytic ane mia)