The mechanism of d-amino acid formation: An alanine racemase from Streptococcus faecalis

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THE MECHANISM OF D-AMINO ACID FORMATION: AN ALANINE RACEMASE FROM STREPTOCOCCUS FAECALIS

BI WILLIS AVERY WOOD Bachelor of Science, 1947 Cornell U niversity

Submitted to the Faculty of the Graduate School in p a r tia l fu lfillm e n t of the requirements for the degree of Doctor of Philosophy in the Department of B acteriology, Indiana U n iversity, February, 1950

ProQuest Number: 10295205

All rights reserved INFORMATION TO ALL USERS The quality o f this reproduction is d e p e n d e n t upon th e quality o f the co p y submitted. In the unlikely e v e n t that th e author did not send a c o m p le te manuscript and there are missing p a g e s, th e se will b e noted, Also, if material had to b e rem oved, a n o te will indicate the deletion.

uest ProQuest 10295205 Published by ProQuest LLC (2016). Copyright o f the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

ACKNOYfLEDGMENT The author takes th is opportunity to express h is gratitu de to Professor I . C. Gunsalus fo r the guidance and a ssista n ce so generously rendered him.

His s c ie n t i f ic enthusiasm has furnished in sp ira tio n and

h is valuable c r itic ism and constant in te r e s t , both academic and per­ son al, has contributed g r e a tly to the d ir e c tio n and execution o f th is work.

VITA W illis Avery Wood was born on August 6, 1921 in Johnson C ity, New York#

He attended the public schools of Binghamton, New York,

graduating from North Senior High School in 1940.

He attended Cornell

U n iv ersity from 1940 to 1943> a t which time he was ordered to a c tiv e duty in the Army o f the United S ta te s , serving in the Quartermaster Corps.

In 1946 he was r e lie v e d of a c tiv e duty and re-entered Cornell

U n iv ersity where he received the degree of Bachelor of Science in 1947* He attended Indiana U n iversity from 1947 to 1950 as a graduate student in B acteriology.

He i s a member of the S o ciety of American B acteriolo­

g i s t s , American Chemical S o ciety , and Sigma X i.

iii

TO ALICE JANE

iv

table; of contents

Page INTRODUCTION ...................................................................................................................

1

METHODS

...........................................................................................................................

4

RESULTS...........................................................................................................................

10

Preparation o f ac e ll- f r e e racemase

..................................................

11

C h aracteristics of alanine racemase

..................................................

12

MECHANISM.......................................................................................................................

15

DISTRIBUTION OF ALANINE RACEMASE...................................................................

20

.

DISCUSSION.....................................................................................................................

22

SUMMARY...........................................................................................................................

26

BIBLIOGRAPHY..................................................................................................................

29

OTHER STUDIES OF MICROBIAL AMINO ACID METABOLISM (rep rin ts appended) The A c tiv ity of Pyridoxal Phosphate in Tryptophane Formation by C ell-Free Enzyme Preparations Function of Pyridoxal Phosphate: R esolution and P u rifica tio n of the Tryptophanase Enzyme of Escherichia c o li Serine and Threonine Deaminases of Escherichia c o l i : vators fo r a Cell-Free System

v

A cti­

INTRODUCTION The lite r a tu r e on the general occurrence, the e s s e n t ia lit y , and the role of D-amino a cid s in b io lo g ic a l systems do not support a u n ifie d view point.

Reports of the re la tio n sh ip of D-amino acids to liv in g systems

have appeared without an adequate knowledge concerning the importance, occurrence, and formation o f th ese compounds being reached.

The data as

w e ll as th e ir in te r p r e ta tio n are c o n flic tin g in many in sta n c e s.

Recent

s tu d ie s, la r g e ly w ith b a cteria , have served to re-emphasize the importance o f D-amino acid s and have prompted the present examination of the mode of formation o f D -alanine. K8gl and Erxleben ( l) increased g r e a tly the in te r e s t in D-amino acids by th e ir report o f high D-glutamic acid content in malignant t is s u e . As high as 15 to 45 per cent of the glutam ic acid is o la te d was reported to be the D-isomer.

Later in v estig a to r s f a ile d to confirm t h is report although

se v e r a l, including Ghibnall and co-workers ( 2 ) , Graph, Rittenberg, and Fos­ te r ( 3 ) , and Wieland and Paul (4) > have found from 1 to 5 per cent of the glutamic a cid is o la te d from normal and malignant t is s u e to be o f the Dform.

These in v e stig a to r s, however, a ttrib u ted the presence o f D-glutamic

acid to racem ization during is o la t io n and therefore attached l i t t l e or no importance to i t s presence. In stu d ies o f microorganisms, D-amino acid s have been reported in e x tr a c e llu la r m aterial, as capsules or in the medium a fte r growth.

The

capsule of B a cillu s anthracis (5* 6 ), and solub le polypeptides secreted by B a cillu s s u b t ilis and B acillu s mesentericus ( 5 ) have been shown to be composed e x c lu siv e ly of D-glutamic a c id .

More recen tly D-amino a cid r e s i­

dues have been reported in several a n tib io tic s including gram icidin (7* £ ) , ty ro cid in (9, 1 0 ), polymyxin ( l l ) , aerosporin (1 2 ), c ir c u lin (13)> and

p e n i c i l l i n (1 4 ). D-amino a cid s have a ls o been reported as su b strates fo r b a c te r ia l growth (1 5 -2 2 ).

In the m ajority of ca ses, e ith e r the s p e c if ic i t y o f the

B-isomer was not shown or the L-isomer or keto a cid analog permitted an equal or greater rate o f growth.

In t h is type of experiment, S n e ll and

Guirard (23) reported DL-alanine to replace vitam in Streptococcus f a e c a l i s . s tr a in R.

fo r the growth o f

Later, S n e ll ( 24 ) showed D-alanine to

be s p e c if ic a lly required and not rep laceab le with the Ir-isomer. a la n in e, however, was replaceable by vitam in B^.

The D-

In lin e with the general

hypothesis that an e s s e n t ia l m etabolite not required for growth i s formed by the organism during growth, S n e ll and Guirard (23) suggested that Dalan in e functions as a precursor fo r the formation of vitam in B.. How6 ever, Bellamy and Gunsalus (25) questioned the v a lid it y o f th is hypothesis because c e lls grown in the presence of D-alanine were nearly devoid o f co­ decarboxylase, although th ey contained the tyrosin e apodecarboxylase which could be a ctiv a te d by vitam in B^ in the form of pyridoxal.

Thus the a lte r ­

nate p o s s ib ilit y , a function of vitam in B^ in the formation of D -alanine, was in d ica ted .

A second in stan ce of apoenzyme formation during growth in

a B d e fic ie n t medium was reported fo r the transaminases by L ich stein , o Gunsalus, and Umbreit ( 26 ) . More rece n tly , Holden, Furman and S n e ll (27 ) 9 and Holden and S n e ll (28) have confirmed the conclusions reached in the enzyme experim ents.

The

a n a ly sis , by m icrob iological a ssay, of c e lls grown in the presence of Dalanine as replacement fo r vitam in B^ showed appreciable amounts o f D-alanine and n e g lig ib le amounts o f the vitam in, whereas c e lls grown with minimal B^ and no D-alanine contained appreciable amounts o f both vitamin B^ and Da la n in e.

This demonstration of a D-alanine requirement for growth and i t s

3 incorporation in to c e l l m aterial represents the f i r s t report o f a D-amino a cid per se a ctin g as an e s s e n t ia l m etab olite. The demonstration th a t vitam in

functions in the formation o f D-

alanine opens the way fo r enzyme stu d ies of the reaction s in volved .

A con­

sid er a tio n o f p o ssib le routes of D-amino acid formation has suggested enzy­ matic racem ization and transam ination (2B) as l ik e ly p o s s ib ilit ie s *

In the

case of racem ization, the D-amino a cid could a r is e by in v e r sio n ,o f the Lisomer, whereas in transam ination i t could be formed from pyruvate with another amino a cid serving as the amino group donor. In the present study, D-alanine was found to a r is e by enzymatic racem ization with vitam in the coenzyme.

in the form o f pyridoxal phosphate serving as

4 METHODS B a c te r io lo g ic a l:

Streptococcus f a e c a l is . s tr a in R (ATCC #8043), p reviou sly

employed fo r stu d ies o f ty ro sin e decarboxylase (2 9 ), and glu tam ic-asp artic transaminase (26) was used.

A ctive racemase preparations were obtained by

growing the c e lls in medium AC.3, which contains 1 per cent each of y ea st extract and tryptone, 0 ,5 per cent dipotassium phosphate and 0*3 per cent g lu co se.

To obtain a large quantity o f c e l l s , 10 l i t e r batches were grown

in 2g-gallon reagent b o t t le s .

The medium was inoculated with a 0 .1 per

cent o f an 8 to 12-hour culture and incubated 10 to 12 hours a t 37° ( f in a l pH, 5*6 to 5*S).

The c e lls were harvested with a Sharpies cen trifu ge, washed

once with water by resuspending in one-tenth the growth volume and cen tri­ fu gin g.

The washed c e l l s were e ith e r acetone dried or dried in vacuo to

y ie ld ly o p h iliz e d preparations* Vacuum dried c e l l s were prepared by suspending the washed c e lls in water to about 10 per cent by weight and drying in vacuo over D rierite to y ie ld about 10 grams o f dry c e l l s per 10 l i t e r s o f medium.

Acetone dried

c e l l s were prepared by p ip ettin g a 10 per cent suspension of c e lls in to 10 volumes of ic e -c o ld acetone with rapid s t ir r in g . on a buchner f i l t e r , washed with acetone

The c e lls were c o lle c te d

and ether; and a ir d ried .

These

preparations racemized 50 to 150 m ic ro liters (jul) of alanine per mg dry wt per hour and were sta b le fo r sev era l weeks i f kept dry or i f stored at -2 0 ° . One micromole (juM) of alan in e i s equivalent to 22.4 p i , Chemical:

D-alanine Determination.

D-alanine was determined manometrically

using a p a r t ia lly p u r ifie d D-amino a cid oxidase (30) •

Racemization of a la ­

nine was carried out in 16 mm t e s t tubes using a 3 ml reaction volume

5 containing the follow in g:

0 .4 ml o f M phosphate b u ffer, pH 8 .1 ; enzyme or

c e l l s , water to 1 .9 ml; and 0 .6 ml of 0 .2 M L -alan in e.

Before ad dition o f

the su b str a te, the tubes were incubated a t 37° fo r 5 m inutes.

The reaction

was allowed to proceed fo r a given time in te r v a l (u su ally 30 m inutes), then stopped by immersing the tubes in b o ilin g water for 5 m inutes.

The protein

or c e l l s were separated by cen trifu g a tio n and a 2 ml a liq u o t removed for D-alanine a ssa y . The D -alanine assay was performed in s in g le sid e arm Warburg fla sk s as fo llo w s:

In the main compartment was a 2 ml sample (equivalent to 1 to

20 /iM of D -alanine); 0 .3 ml o f M phosphate b u ffer, pH 8 .1 ; and 0 .2 ml of w ater.

The sid e arm contained 0 .5 ml of D-amino a cid oxidase; and the

center w e ll, 0.15 ml o f 20 per cent potassium hydroxide. A fter 10 minutes o incubation a t 37 , the D-amino acid oxidase was tipped and the oxidation follow ed for one hour.

Added samples o f D-alanine up to 20 pM. were com­

p le t e ly oxidized w ithin 30 minutes in the presence o f the reaction mixture or c e l l suspension.

Since the D-amino a cid oxidase preparation contained

c a ta la se , J jxM oxygen uptake corresponds to 1 pM o f D -alanine.

Therefore,

the p.1 o f oxygen taken up tim es 2 equals the p i of D-alanine present in the sample. In order to determine racemase a c t iv it y more conveniently, a d ir e c t manometric assay for D-alanine formation using an excess of D-amino a cid oxidase was employed, based on the follow ing rea ctio n s:

6 Racemase L-alanine

D-alanine

a)

Pyruvate 4

(2)

B alPO,# 6 4 D-amino a cid D-alanine 4 0, 2

4*oxidase ca ta la se

(3)

(in oxidase)

L-alanine +

\

CL

Pyruvate

4

NH^

4

H^O

(4)

Thus the yul of oxygen taken up m u ltip lied by 2 i s equivalent to the ;ul of D-alanine formed.

Under the conditions used and with an excess of D-amino

a cid oxidase, the rate lim itin g step i s the racem ization so that the race­ mase a c t iv it y i s proportional to the rate of oxygen uptake. The d ir e c t assay was performed in double sid e arm Warburg cups with additions as fo llo w s:

In the main compartment, 0 ,3 ml of M phosphate b u ffer,

pH 8 .1 ; c e lls or enzyme, and water to 1 .6 ml; in the center w e ll, 0.15 nil of 20 per cent potassium hydroxide; in one sid e arm, 0 ,4 ml o f 0 .2 M L-alanine (80 juM); and in the second sid e arm, 1.0 ml of concentrated D-amino acid oxid ase.

The cups were incubated 5 minutes a t 37 °> both sid e arms tipped

sim ultaneously and the rea ctio n allowed to proceed 10 minutes before the cocks were c lo se d , then the rate of oxygen uptake was measured over a 30minute period.

The method was standardized, using both graded le v e ls of

c e lls and o f a c e ll- f r e e enzyme.

As shown in figu re 1, the rate of race­

m ization was e s s e n t ia lly proportional to enzyme concentration up to 760 alL of alanine racemized per hour (oxygen uptake equivalent to 760/2/11 or * B^alPO^ and B^NH^POi denote pyridoxal phosphate and pyridoxamine phosphate r e sp e c tiv e ly throughout the t e x t .

j x L D -A LA N IN E FORMED /H O U R

o K>

> o

Q

g

O)

H O

z o (/> \

00

CJ 2

r

o

& &

"0 o 2 n

3 F

rn w

(a> ?

1000

o

7 360 jul per hour).

Dried c e l l s a t le v e ls o f 125 to 2000 Y racemized alanine

equivalent to 10 to 160 >ul o f oxygen uptake per hour (20 to 320 ;ul of alanine formed). DL-alanine:

T otal alanine was determined c o lo rim etrica lly by a

m od ification o f the method o f Aquist (31)•

The alanine was f i r s t converted

to l a c t i c acid by n itrou s a cid , and the la c ta te measured co lo rim etrica lly by the method of Barker and Summerson (3 2 ).

Using the Evelyn colorim eter

and a 565 mja f i l t e r , the o p tic a l d en sity was proportional to alanine con­ cen tra tio n from 10 to 100*/. Pyruvate:

Pyruvate was determined by measuring the oxidation of

DPNH^ ^ a t 340 mp. in the Beckmann spectrophotometer using a b eef-heart la c ­ t i c dehydrogenase, p u r ifie d by the method of Straub (3 3 ). Pyridoxal Phosphate:

Pyridoxal phosphate was assayed using a 2 p u rified apotyrosine decarboxylase from S. fa e c a lis prepared e s s e n t ia lly by the method o f Epps (34) •

The rate o f tyrosin e decarboxylation (v) was

measured in the presence of the sample, and a lso with an excess of pyridoxal phosphate which was assumed to give a decarboxylation rate not g rea tly d if ­ feren t from the th e o r e tic a l maximum v e lo c ity Of ). max . pyridoxal phosphate d isso c ia tio n constant, K »

Using the ty ro sin e—S 1.5 x 10 m oles/ l i t e r

(3 5 ), and the Michaelis-Menten equation (3 6 , 37 )> ihe concentration (c) of pyridoxal phosphate in the sample was calculated as in the follow ing case where v (from 1 ml of D-amino acid oxidase per cup) equals 100 /ll/h r ^ DPNH and DPN denote the reduced and oxidized forms of diphosphopyridine n u cleo tid e. 2 Kindly furnished to us by Mr. L. I . Feldman of th is laboratory.

8

and the assumed V i s 570 M l/hr. max.

v

—

V max.

* c

so lv in g fo r cs

c

=

K . v m V +■ max.

c

=

v

1 .5 x 10“

8

. 100

570 + 100 c

=

2 .2 x 10

-9

m o le s /lite r

or

1*0 milligamma/ ml D-Amino Acid Oxidase:

D-amino acid oxidase was concentrated and

p a r t ia lly p u rified with s lig h t m odification from the method of Negelein and BrSmel (3 0 ).

Pig kidney was d ecorticated , homogenized, added to acetone

a t -2 0 ° , and the p recip ita ted tis s u e then f ilt e r e d and d ried . the enzyme, 100 grans of the dry powder was

To extract

stir r e d with 500 ml o f M/60

pyrophosphate b u ffer, pH 8 .3 , a t 37° for 1 hour and then centrifuged.

The

supernatant was brought to pH 5.1 with d ilu te a c e tic a cid , the temperature o quickly ra ised to 37 } held for 5 minutes and then cen trifuged . The super­ natant then was brought to 40 per cent saturation with s o lid ammonium su l­ fa te and centrifuged; the p r e c ip ita te was red issolved in 100 ml of M/l5 pyrophosphate b u ffer and held a t -1 4 ° u n til used.

Oxidase a c t iv it y varied

between 35 and 165 Ml o f oxygen uptake per 5 minutes per 0*5 ml of enzyme under conditions o f the D-alanine assay described above.

In order to use

9 t h is preparation fo r assa y o f the resolved racemase, i t was necessary to know i t s pyridoxal phosphate concentration.

For th is purpose the pyridoxal

phosphate assay using apotyrosine decarboxylase was performed as above and the oxidase found to be e s s e n t ia lly free of th is coenzyme sin ce 1 ml of the enzyme contained approximately 3 milligamma.

This le v e l o f coenzyme

has a n e g lig ib le e f f e c t upon the rate of racem ization. L-Alanine:

L-alanine was prepared by asym etric hydrolysis of

D L-acetylalanine using a p a r t ia lly p u rified L-carboxypeptidase preparation from p ig kidney according to the method of Fodor, P r ic e, and Greenstein (3&) •

The D-alanine con ten t, as determined manometrically with D-amino

a c id oxidase, was approximately 0*5 per cent. Reduced Diohosphoo.vridine N ucleotide;

DPNH^ was prepared from

Schwartz cozymase (60 per cent p u rity) by reduction with sodium hydrosul­ f i t e and p r e c ip ita tio n from methanol with ethanol and ether e s s e n t ia lly by the method of Ohlmeyer (39)*

10 RESULTS The in d ic a tio n of a fu nction o f vitam in B/ in D-alanine formation 6 has led to a study o f the enzyme involved in th is p rocess. From the knowledge a v a ila b le o f the fu n ction of vitamin B^, a transaminase s p e c if ic fo r the B- rather than the L- isomer of alanine seemed p o ss ib le , in which case pyruvate would be a precursor of D -alanine.

The p o s s ib ilit y of L-

alan in e being the precursor of D-alanine was a ls o considered, e s p e c ia lly in view of S n ell* s suggestion (2 8 ).

In the l a t t e r case, however, i t seemed

th at the mechanism might in volve the in te r a c tio n of two transaminases lin k ed by a c a r r ie r , as in the case of the asp a rtic-a la n in e reaction (AO, 4 1 ), Experiments designed to study the con ditions necessary fo r D-alanine formation were undertaken with dried c e l l preparations o f S. f a e c a lis . stra in R, harvested from neutral medium.

Such c e lls were se le c te d because they

contained good a c t i v it y for other amino a cid reaction s e s p e c ia lly the trans­ aminases ( 26 ) .

In th ese stu d ies i t was found that D -alanine, which was

added as the only su b stra te, disappeared under aerobic con d ition s.

Subse­

quently i t was found th a t with L-alanine as the su b stra te, D-alanine was formed.

Thus an enzyme or enzymes which racemize alanine were presen t.

The mechanism, however, could not be determined by th is means. The p o s s ib ilit y , in d icated by growth experiments of vitamin

in

the form of pyridoxal phosphate actin g in th is reaction , was te ste d by measuring the rate o f L-alanine racem ization in the presence and in the ab­ sence of pyridoxal phosphate.

As shown in figu re 2, pyridoxal phosphate

stim ulated the rate of alanine in v ersio n . The mechanism of racem ization was not im p lic it from th ese experi­ ments, sin ce c a ta ly tic amounts of another substrate or a carrier present in the c e lls could serve as an interm ediate in the rea ctio n .

Therefore,

Figure 2.

Stimulation of alanine racemization

by pyridoxal phosphate.

Conditions as in Figure

1 except with 5 mg dry wt of c e lls and pyridoxal phosphate as indicated#

ALANINE RACEMASE

200

DRIED CELLS - S. FAECAL IS 175

D-ALANINE

FORMED

150

125

100

50

25

0

5

10

15 MINUTES

20

25

30

11 c e l l - f r e e preparations were obtained, p a r tia l p u r ific a tio n undertaken and the properties of the enzyme(s) studied to determine the mechanism and the ro le o f pyridoxal phosphate. Since the reaction as measured amounted to a racemase action regard­ le s s o f the mechanism, the sim p lest case was to assume a sin g le enzyme capable o f racemizing alan in e in the presence of pyridoxal phosphate as the coenzyme.

Therefore, u n til a more complex mechanism can be shown, the en­

zyme i s referred to as alanine racemase.

The a c t iv it y was measured by the

rate of D-alanine formation in the continuous assay described in the methods s e c tio n . i

Preparation o f a C ell-Free Racemase Five grams o f vacuum dried c e lls and 2*5 grams o f f in e ly powdered carborundum were suspended in 50 ml o f 0 .1 M phosphate b u ffer, pH 8 .1 , and subjected to sonic o s c illa t io n in a Raytheon 50-watt, 9 K.C. m agnetrostrictio n o s c illa t o r fo r 1 hour a t 8 ° .

The debris was removed by cen trifu gation

a t 18,000 RPM in the In tern a tion al cen trifu ge high-speed head (28,000 G.) for 15 m inutes. was

A fter standing overnight in the r e fr ig e r a to r , the debris

resuspended in 50 ml of 0 .1 M phosphate b u ffer, subjected to sonic

o s c illa t io n fo r i j hours and cen trifu ged .

The

combined supernatants con­

tained 96 per cent o f the a c t iv it y present in the dried c e l l s .

The enzymes

were p recip ita ted with ammonium s u lf a t e , adsorbed on calcium phosphate g e l, and elu ted with phosphate b u ffer. shown in the flow sh e e t.

The d e ta ils of these operations are

For each step in the

process, the degree of

reso lu tio n with respect to pyridoxal phosphate and the p u rifica tio n obtained are shown in Table I .

546724

FLOT SHE ST FOR THE PURIFICATION OF ALANINE RACEl'ASE Suspend 5 K®* vaouum «dried o e l l s (15,200 u n i t s ) and 2*5 gm. powdered carborundum (?500) In 50 “ l* 0*1 U phosphate b u f f e r , pH 0*1) s u b je o t to so n ie o s c i l l a t i o n 1 h r* a t 8°} c e n tr if u g e

¥

Resuspend i n 50 ml* 0*1 M phosphate b u f f e r , pH 8 .1 , s u b je o t to so n ie o s e i l l a t i o n l£ h r s . , o e n trifu g e C e il d e b ris • d is c a r d

Combine s u p e rn a ta n ts , (ll;,5 5 0 u n i t s ) Add s o li d (NHi ) ^ 0 t o lOOji s a t u r a t i on, c e n t r Ifu g e 1 D iscard

D isso lv e in 100 m l. 0 .1 11 phosphate b u f f e r , pH 8 .1 , add s a tu r a te d (NIL )^50i t o h&A s a tu r a tio n ; o e n trifu g e ri— 1

r

D iscard

li D isso lv e in 50 “ I* 0 .1 1! phosphate b u f f e r , pH 8 .1 (5,700 u n i t s ) ; add s a tu r a te d (NHjJoSOr to 50$ s a t u r a t io n , c e n tr i f uge 1

II

1 Add s a tu r a te d (NH^)gSCS to 75$ s a tu r a tio n ; o e n trifu g e 1 1 D iscard

1 Add s a tu r a te d (NHi JoSCh t o 66% s a tu r a tio n ; c e n trifu g e

D isc ard

1

II D issolve i n 50 ml* 0 .1 11 phosphate b u f f e r , pH 8 .1 (5,160 u n i t s ) , add s a tu r a te d to 56$ s a tu r a tio n , c e n trifu g e 1

I

D iscard

D iscard

Add s a tu r a te d (SHk J^ O ^ t o 70$ s a tu r a tio n , o e n trifu g e 1

n

D iseard

D isso lv e in 25 m l. 0 .1 1! phosphate b u f f e r , pH 8 .1 (2,800 u n i t s ) , add w ith s t i r r i n g 3*58 6 ° . CajtPC^Jp g e l, c e n tr if u g e

II

n

1

“ 1 D iscard

Wash w ith fo u r 50 ml* p o rtio n s o f w ate r; c e n tr if u g e

1

E lu te w ith 25 ml* M phosphate b u f f e r , pH 8 *1 , s t i r 2 hours in th e e o ld , o e n trifu g e

I____________________________ D iscard

D iscard washings

1

A lanine ra o e a a s e , 25 n l* , 2,100 u n i t s , 1J $ reo e v ery 60^ re so lv e d

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o

oco

o H* H H P d~ H*

B

•d ^yv*3 O co

©

P

s

B

tr (I) qq Cj-'g

©

3-

©

* p 3 HPC © P CO

3

H HsH* O

• o H* ©

P

c+ H* O 3

VJ-T

Vl O

vO

V5 *

rv

VO

•

12 As p rev io u sly mentioned, d isin te g r a tio n o f the c e lls by sonic o s c illa t io n r e le a se s nearly 100 per cent o f the enzyme.

This procedure,

however, a ls o y ie ld s ex tra cts of unusually high protein con ten t.

Pre­

c ip ita tio n w ith ammonium s u lfa te removed much of the in a ctiv e p rotein, but a ls o r e su lted in a su b sta n tia l lo s s in enzyme a c t iv it y .

In ad d ition ,

th is procedure did not further resolve the already p a r tia lly resolved enzyme.

A b e tte r procedure fo r the fra ctio n a tio n o f the enzyme has not

as y et been found.

Adsorption of the enzyme on calcium phosphate g e l and

e lu tio n with phosphate buffer resu lted in an in crease in p u rity without great lo s s o f the enzyme.

The o v e r a ll procedure y ield ed 14 per cent of

the a c t i v it y present in the dried c e l l s , 60 per cent reso lu tio n with respect to pyridoxal phosphate, and a p u r ific a tio n (based upon p rotein content) of about fo u rfo ld .

Other enzyme preparations, obtained by only s lig h t m odifi­

ca tio n of th is procedure, were as much as 80 per cent resolved .

The enzyme o

was sta b le fo r several weeks when stored in the refrig era to r a t 4 ♦ Ex­ periments designed to show whether or not p u r ific a tio n had decreased or elim inated the le v e ls of p o ssib le interm ediates and in te r fe r in g enzymes are described in the follow ing s e c tio n .

C h aracteristics of Alanine Racemase The p u rified racemase catalyzed the transformation of e ith e r isomer of alanine to the racemic mixture in the presence of coenzyme and the s p e c ific su b stra te.

The data are shown in fig u res 3 and 4 .

The t o t a l a la ­

nine concentration remained constant as indicated by s p e c ific colorim etric a n a ly sis a fte r conversion to la c t ic a cid with nitrous a c id . phosphate requirement i s shown a ls o .

The pyridoxal

I t seemed lik e ly that other reactants

mM

d -a la n in e /m l.

o

r\>

10

RACEM ASE

o

ALANINE

3 —

13 should be removed through the p recip ita tio n steps of the p u rifica tio n pro­ cedure and thus that a d ir ec t racemization i s involved in the reaction . Other evidence on th is point i s presented in the mechanism section* In order to a scerta in the pH of optimum a c t iv it y , enzyme a c t iv it y was te ste d a t various pH in ter v a ls between 5*3 and 8 .7 .

Since D-amino acid

oxidase does not function a t an acid pH, the tube assay described in the methods sec tio n was used.

As shown in figure 5* the optimum pH i s above 8 ,

suggesting th a t the unionized amino group i s required for a c t iv it y . The influence of the Lr-alanine concentration upon the rate of race­ mization i s shown in figure 6 .

The enzyme a f f in it y for the substrate is

rather low as indicated by the lack of substrate saturation at SO juM of D-alanine per 3 ml.

The Michaelis-Menten constant (37) i s about 8*5 x 10

moles per l i t e r , equivalent to 25 pM. per 3 ml*

-3

The s p e c if ic it y of the en­

zyme for alanine as the substrate was indicated by the lack of racemization o f 4CO to O H O o H

C+ O O a o

P co P

O P p a

OP

PCO 3

ft o

p h* H * CO *& >

>c

B

-j O'

VjO

o oCO

O H O CO c+ P H* a H*

O' -3

H

f &

3

4o ft

CD

i~ 3

O O

p o H* H £CO CO

PJ CO CD

P

a § § g C O

£CT c+ H j H* £ p CO 4

II

oP o a* P

> CO *T3 ft

O c3 p

ft

a

s

CO

oft p CO

o VjJ H to >c

HI V

8

H -F~ O

p p

H*

P

ft

a a c t iv it y using conditions described in figure 1 .

Measurement of the rate

of oxygen uptake eith er fa ile d to show the presence of a racemase or led to inconclusive r e s u lts , as in the case of liv e r and kidney, due to a high endogenous resp iration .

22 DISCUSSION An enzyme has been described which forms a D-amino acid, s p e c ifi­ c a lly D -alanine•

From the fractionation procedure and the ch aracteristics

of the reaction, i t appears that one enzyme u tiliz in g pyridoxal phosphate as a coenzyme is involved.

In analogy to the chemical mechanism of amino

acid racemization, an explanation of the function of the enzyme and the coenzyme, and knowledge of the mechanism of th is process is desirable. In i t s sim plest form, the present theory for chemical racemization of amino acids i s written as a tautomeric s h if t of the hydrogen attached to the *-carbon as follow s: I NH

H I -c I NH | I

.J>

c

. — ■""

OH

r J * -c = = c I OH NH | II

. >

L —c I H

J>

c;

OH III

The migration of hydrogen and the formation of the double bond destroys the asymmetry of the molecule (II) which may on ketonization revert to the L- or D-configuration as represented by I and I I I . The experiments of Bergraann ( 56, 57) and du Vigneaud and Meyer ( 58 ) show that amino acids are rapidly racemized in the presence of acetic anhydride.

These investigators suggest that the mechanism of the process

involves ring formation of the azlactone type (III) followed by tautomeric s h ift of hydrogen as indicated in the following diagram:

H— CH

< ft— CH 'OH

HH

ft— CH---------------C=0 OH V /0

NH 1—0 C I ch3

1

II CH, ^

n

h i

ft

C = = C —OH NW

I

° I CH3 ^

iv

23 E sse n tia lly the process occurs by N-acetylation followed by ring closure by dehydration in the presence of ca ta ly tic amounts of acetic anhydride* By analogy, i t i s suggested that enzyratic racemization may occur by a sim ilar type of ring formation in which pyridoxal phosphate is in­ volved as follow s: ,0 CH— CH — c ; OH NH,

VH ch2opo3 h2

CH 0P0o Ho 2 3 2

HO CH. 3 Y

o-quinone form, II

aldehyde form, I

pyridoxal phosphate

CH— CH— C;

H-C-OH CH^OPO^H^ + enzyme

CHr-CH

C-OH

C=0

1 I

HO J

\ ch2opo3 h2

NH

HO ^ ^ C H jjOPO^ CH,

CH, IV

0 C^KIH

V V

24 I t i s to be noted that in th is case participation of a carbonyl or orthoquinone group is suggested rather than a carboxyl group which acts in chemical racemization.

The condensation between aldehydes and amines i s

a general reaction for the formation of azomethines or S ch iff’s bases. Sim ilarly, 1 ,4 addition b etw een * ,/? , unsaturated ketones and amines i s w ell known.

The orthoquinone (II) form of pyridoxal phosphate may be con­

sidered to be a n * , p unsaturated ketone since i t contains the grouping: &

H— C — OH

Gulland and Mead (59) studied a sim ilar condensation of aromatic aldehydes with D-phenylalanine and suggested that the condensation product may be of the type:

NH

I

H -C -O H I Ar This compound i s id e n tic a l with the type postulated for condensation be­ tween pyridoxal phosphate and alanine ( I I I ) * The function of the enzyme in racemization i s not known, but pre­ sumably i t would function by favoring enolization or ring closure in analogy to the decrease of energy of activation by ring formation in pep­ tid e hydrolysis as outlined in the hypotheses of Smith (60, 6l ) .

Ring

closure of compound III would form a 2 , 3 -dihydroazlactone or 5-oxazolidone.

25 At the moment, precise evidence for the mechanism of racemization or the mode of action of pyridoxal phosphate in any of the enzymatic re­ actions where i t i s required i s not availab le.

However, indications of

the properties as regards complex formation (and condensation) are a v a il­ able from s h ifts in the u ltr a v io le t spectrum upon addition of various amino acids ( 62 )*

26

SUMMARY In a study of D-amino acid formation in Streptococcus fa e c a lis. i t has been found that D-alanine is formed from L-alanine.

Investigation

of the process with dried c e lls and a p artially purified enzyme revealed that both D- and L-alanine were converted to the raceme mixture.

The

process was demonstrated to be a racemization by measuring the increase or decrease in D-alanine concentration equal to one-half the isomer added while the to ta l alanine concentration remained constant* A method for measuring racemase a ctiv ity was devised employing a manometric measurement of D-alanine formation with D-amino acid oxidase. For routine assays L-alanine was used as substrate in the presence of excess D-amino acid oxidase.

Under these conditions the rate of oxygen

uptake is proportional to the rate of conversion of L-alanine to D-alanine. To study the mechanism of racemase action, the enzyme was extracted from the dried c ells by sonic oscillation and purified by precipitation with ammonium su lfate, adsorption on calcium phosphate gel and elution with phosphate buffer.

This procedure yielded a racemase which was 60

per cent resolved and purified about fourfold. Using the purified enzyme, the rate of racemization was measured at various pH intervals between 5*3 and 8*7,—optimum pH above 8 . Substrate sp e c ific ity was tested by measuring the rate of racemiza­ tion of the L-isomer of various aliphatic, heterocyclic and aromatic amino acids.

None of the amino acids tested including*-aminobutyric acid were

racemized.

Thus the enzyme appears to possess a high substrate specificity*

Both the dried ce lls and the partially purified enzyme require pyridoxal phosphate for maximum a c tiv ity .

Pyridoxamine phosphate is

27 without coracemase a c tiv ity . Studies on the mechanism of racemase action failed to indicate a transaminase function in the reaction.

Transamination between pyruvate and

a series of amino donors including pyridoxamine phosphate did not yield alanine. An assay of representative species of bacteria grown under various conditions shows the racemase to have a general occurrence in bacteria. One mold and one yeast strain tested were without a ctiv ity ,

A similar

assay of extracts of rabbit tissue failed to show appreciable racemase a c tiv ity .

BIBLIOGRAPHY

29 BIBLIOGRAPHY

1.

Kttgl, F., and Erxleben, H., Z. physiol, chem., 2^8, 57 (1 9 3 9 ).

2.

Chibnall, A. C., Rees, M. W., Williams, E. F., and Boyland, E., Biochem. J ., XL> 285 (1940).

3.

Graff, S ., Rittenburg, D., and Foster, G. L., J. Biol, Chem,, XXL 745 (1940).

4.

Wieland, T., and Paul, W., Ber. dtsch. chem. Ges., 77, Abt. B, 34 (1944).

5.

Ivanovics, G., and Bruckner, V., Z. Immun. Forsch., $0, 304 (1937).

6.

Hanby, W. E., and Rydon, H. N., Biochem. J ., /£ , 297 (1946).

7. Christensen, H. N., J. Biol. Chem., 1£L, 319 (1943). 8. Christensen, H. N., J. B iol. Chem., 154. 427 (1944). 9. Hotchkiss, R. D., J. Biol. Chem., 141, 171 (1941). 10. Gordon, A. H., Martin, A, J. P ., and Synge, R. L. M., Biochem. J ., . X L 313 (1943). 11.

Cosden, R., Gordon, A. H., Martin, A. J. P., and Synge, R. L. M., Biochem. J ., 1*1, 590 (1947).

12. B ell, P. H., Bone, J. F., English, J. P ., Fellows, C. E., Howard, K. S ., Rogers, M. M., Shepherd, R. G., Winterbottom, R., Dornbush, A. C., Kushner, S ., and SubbaRow, I . , Ann. N. Y. Acad. S c i., X L ^97 (1949). 13. Peterson, D. H., and Reineke, L. M., J. B iol. Chem., 181, 95 (1949). 14. du Vigneaud, V., Carpenter, F. H,, Holley, R. W., Livermore, A. H., and Rachele, J. R., Science, 104, 431 (1946). 15.

Konikova, A. S ., Azarkh, R. M,, and Dobbert, N. N., Biokhimiya, 10, 82 (1945).

16. Webster, M. D., and Bernheim, 17. Fling, M., and Fox, S.

W., J,

F., J . Biol. Chem., 114, 265 (1936). Biol. Chem., 160, 329 (1945).

18. Baugness, L. C., J. Bact., 32, 299 (1936). 19. Woods, D. D., and Clifton, C. 2 0.

E ., Biochem. J ., ^ 2 , 345 (1936).

Camien, M. N ., and Dunn, M. S ., J . Biol. Chem., 179, 935 (1949).

30 21.

Hegsted, D. M., J . B iol. Chem., l£ k 741 (1945).

22.

Gause, G.

F., and Smaragdova, N. P ., B iol. J. Russ., £, 399 (1938).

23*

Snell, E.

E., and Guirard, B. M., Proc. Nat. Acad. S ci., 2£, 66 (1943).

24.

Snell, E.

E., J. B iol. Chem., 1£8 , 497 (1945).

25.

Bellamy, W. D., and Gunsalus, I. C., J. Bact., ^0, 95 (1945).

26.

Lichstein, H. C., Gunsalus, I . C., and Umbreit, W. W., J, Biol. Chem., 161 , 311 (1945).

27.

Holden, J. T .. Furman, C., and Snell, E. E., J. Biol. Chem., 178, 789 (1949).

28.

Holden, J. T., and Snell, E. E., J. B iol. Chem., 178, 799 (1949).

29.

Gunsalus, I. C., and Bellamy, W.D., J. B iol. Chem., 155. 357 (1944)•

30.

Negelein, E., and Brttmel, H., Biochem. Z., 300. 225 (1939).

31.

Aquist, H. E. G., Acta, Physiol. Scand., 1^, 297 (1947)*

32.

Barker, S. B., and Summerson, W.H., J. Biol, Chem., 138. 535 (1941).

33*

Straub, F. B., Biochem. J .,

34*

EpPs j H. M. R., Biochem. J ., ^8 , 242 (1944).

35*

Gunsalus, I . C., and Umbreit, W.W., J. Biol. Chem., 170 . 415 (1947).

36 .

Michaelis, L ., and Menten, M. L., Biochem. Z., 4.2, 333 (1913).

37.

Lineweaver, H., and Burke, D., J. Am. Chem. Soc., j?6 , 658 (1934).

38.

Fodor, P. J ., Price, V. E., and Greenstein, J. P., J. Biol. Chem., 178. 503 (1949).

39.

Ohlmeyer, P. Biochem. Z., 297. 66 (1938).

40 .

Kritzman, M. G., Biokhimiya,

41 .

O'Kane, D, E,, and Gunsalus, I . C., J. Biol. Chem., YfO, 433 (1947).

42.

Wood, W. A.. Gunsalus, I . C., and Umbreit, W. W., J. Biol. Chem., 170. 313 (1947).

43 .

0 1Kane, D. E., and Gunsalus, I. C., J. Biol. Chem., 120, 425 (1947)*

44 .

Umbreit, W. W., 0'Kane, D. J ., and Gunsalus, I . C., J. Biol. Chem., 176. 629 (1948).

483 (1940).

691 (1939).

31 45* Green, D. E., Leloir, L. F., and Nocito, V., J. Biol. Chem* 161, 559 (1945). 46 .

Kritzman, M. G., and Samarina, 0 ,, Mature, 158. 104 (1946).

47* Snell, E. E., J. Am. Chem. Soc., 62 , 194 (1945). 48.

Ames, S. R., Sarma, F. S ., and Elvehjem, C. A., J. Biol. Chem., 167. 135 (1947).

49* Umbreit, W. W., O'Kane, D. J ., and Gunsalus, I . C., J. Bact., jil, 576 (1946). 50.

Bellamy, W. D., Umbreit, W. W., and Gunsalus, I . C., J. B iol. Chem., 160. 461 (1945).

51.

Snell, E. E., Personal Communication.

52.

Jagannathan, V., and Luck, J. M., J. Biol. Chem., 179. 561 (1949).

53.

Jagannathan, V., and Luck, J. M., J. Biol. Chem., 179. 569 (1949).

54* Sutherland, E. W., Cohn, M., Posternak, T,, and Cori, C. F., J. Biol. Chem., 180, 1285 (1949). 55.

Stanier, R. I ., J. Bact., Jj£, 477 (1948).

56 .

Bergmann, M., and KiSster, H., Z. physiol, chem., 159. 179 (1926).

57.

Bergmann, M., and Zervas, L., Biochem. Z,, 203 . 280 (1928).

58.

du Vigneaud, V., and Meyer, C, E., J. Biol. Chem., ,22* 143 (1932-33).

59.

Gulland, J. M., and Mead, T. H,, J. Chem. Soc., 210 (1935).

60.

Smith, E. L,, Proc. Nat. Acad. S c i., 22, 80 (1949).

61.

Smith, E. L., Federation Proc., 8, 581 (1949)*

62.

Wood, W. A., and Gunsalus,

I.

C., unpublished data.

OTHER STUDIES OF MICROBIAL AMINO ACID METABOLISM

T H E A C T IV IT Y O F P Y R ID O X A L P H O S P H A T E I N T R Y P T O ­ P H A N E F O R M A T IO N B Y C E L L -F R E E E N Z Y M E P R E P A R A T IO N S

B y

W . W . U M B R E IT , W . A . W O O D , and

I. C . G U N S A L U S

(F rom the L aboratory o f B acteriology, College o f A g ric u ltu re , C ornell U n iv e rsity , Ith a ca )

R

e p r in t e d

pr o m

T H E

JO U R N A L

O F B IO L O G IC A L C H E M IS T R Y N o . 2 , O c t o b e r , 1946

V o l. 165,

M a d e in U n ited S ta les of A m erica

R e p rin te d fro m T h e J o u r n a l o f B io l o g ic a l C h e m is t r y V ol. 105, N o . 2, O c to b e r, 1940

T H E A C T IV IT Y OF P Y M D O X A L P H O SP H A T E I N T R Y P T O ­ P H A N E F O R M A T IO N B Y C E L L -F R E E E N Z Y M E P R E P A R A T IO N S* Sirs: I t has been known for som e tim e th a t the tryptophane requirement of certain bacteria can be supplied b y indole .1 M ore recently T atum and Bonner , 2 using a strain of Neurospora, have demonstrated th e synthesis of T ryp to p h a n e F orm ation by Cell-Free Enzym e P reparation In d o le lo s t E n zy m e from

A d d itio n s

T rg p to io rm ed

M icrom oles X 10s, 1 h r.

F re sh fro z e n m y c e liu m

+ +

M y c e liu m h e ld 7 days

W + + + W + + +

M y c e liu m h e ld 32 d ay s

W + + W + +

0 . 4 4 juM i n d o l e 0 .4 4 “ “ + 1 dl- s e r i n e ith o u t a d d itio n s 0 . 4 3 ju n c i n d o l e 1 m g . d Z -s e rin e 0 . 4 3 ju m i n d o l e + 1 d Z -s e rin e ith 10 y p y rid o x a l p h a te * 0 . 4 3 a im i n d o l e 1 m g . d Z -s e rin e 0 . 4 3 a im i n d o l e + 1 d Z -s e rin e ith o u t a d d itio n s 0 . 4 2 ju m i n d o l e 0 .4 2 “ “ + 1 d Z -s e rin e ith 10 y p y rid o x a l p h a te * 0 . 4 2 >uM i n d o l e 0 .4 2 “ “ + 1 d Z -s e rin e

In d o le lo s t

T ry p to ­ phane form ed

M icrom oles X 10s, 2 h rs .

3

2

5

4

32

34 0 0 0

44

46 0 0 0

6

13

18

21

3

0 0 0

0

0 0 0

14

19.

31

34

4

2

0

1

5

5

6

6

0

0

0

0

19

20

32

40

m g.

0

0

m g. p hos­

m g.

m g. phos­

m g.

* 1 0 y o f t h e b a r i u i n s a l t o f p y r i d o x a l p h o s p h a t e , S a m p le 5 0 - 4 .6

tryptophane from indole and serine. From this mold we have been able to prepare a cell-free system which converts indole plus serine into trypto­ phane. The enzym e has been resolved and pyridoxal phosphate found to * S u p p o rte d in p a r t b y a g ra n t fro m th e N u tr itio n F o u n d a tio n , In c . 1 F i l d e s , P . , B rit. J . E x p . P a th ., 2 2 , 2 9 3 ( 1 9 4 1 ) . S n e l l , E . E . , Arch. Biochem., 2 , 3 8 9 (1 9 4 3 ). 2 T a t u m , E . L . , a n d B o n n e r , D . , Proc. N a t. A ca d . S c ., 3 0 , 3 0 ( 1 9 4 4 ) . 731

732

L E T T E R S TO T H E E D IT O R S

restore th e activ ity . W ith th e cell-free enzyme, indole disappearance3 was found to correlate w ith try p to p h a n e form ation.4 T he cell-free enzym e m ay be prepared from the m ycelium of Neurospora sitophila grown for 3 days, washed w ith distilled w ater, frozen, homogenized in 1.5 tim es its weight of 0.1 m phosphate buffer a t pH 7.5, an d centrifuged; 0.5 ml. of this prep aration ( = 5 mg. of protein), m ade up to 1 m l. w ith addi­ tions, was incubated a t 37° (see the table). If th e frozen mold tissue is held several days before hom ogenization, the enzyme preparations obtained are m uch less active, b u t m ay be re­ activated by th e addition of pyridoxal p hosphate.6 Since pyridoxal phosphate does n o t influence try p to p h an e form ation in th e absence of either serine or indole, its action appears to be in th e indole-serine system leading to tryptophane. These d ata provide evidence of another function of pyridoxal phosphate in addition to its role in am ino acid decarboxylation6 and in transam ina­ tio n .7 The relationship of this function to the action of vitam in B6 in try p to p h an e m etabolism rem ains to be determ ined.8 Laboratory of Bacteriology College of Agriculture Cornell University Ithaca

W. W. U m b r e i t W. A. W o o d I. C. G u n sa l u s

R eceived for p u b licatio n , A ugust 17, 1946 3 S tan ley , A. R ., and S pray, R . S., J . B a d . , 41, 251 (1941). 1 Snell, F . D ., and Snell, C. T ., C olorim etric m eth o d of an a ly sis, New Y o rk , 2, 211 (1937). 5 G unsalus, I. C ., U m b reit, W. W ., B ellam y, W. D ., an d F o u st, C. E ., J . Biol. Chem., 161, 743 (1945). 0 G unsalus, I. C., B ellam y, W. D ., and U m b reit, W. W ., J . Biol. Chem., 155, 685 (1944). B rad d ily , J ., and G ale, E . F ., Nature, 155, 727 (1945). U m b re it, W. W ., and G unsalus, I. C., J . Biol. Chem., 159, 333 (1945). 7 L ichstein, II. C., G unsalus, I. C ., and U m b reit, W. W ., J . Biol. Chem., 161, 311 (1915). G reen, D . E ., Leloir, L. F ., an d N ocito, V., J . Biol. Chem., 161, 559 (1945). 8 N u tr . Rev., 3, 72 (1945).

R e p rin te d fro m T h e J o u r n a l o f B io l o g ic a l C h e m is t r y V ol. 170, N o . 1, S e p te m b e r, 1947

F U N C T IO N OF P Y R ID O X A L PH O SP H A T E : R ESO L U TIO N A N D P U R IF IC A T IO N OF T H E T R Y P T O P H A N A S E E N Z Y M E OF E SC H E R IC H IA COLI B

y

W . A . W O O D ,* I . C . G U N S A L U S , a n d W . W . U M B R E I T

cF rom the L a b o ra to ry o f B a cterio lo g y , C ollege o f A g r ic u ltu r e , C o rn ell U n iv e r s ity , Ith a c a ) (R e c e iv e d fo r p u b lic a tio n , J u n e 2 , 1947)

Since H opkins and Cole ( 1 ) dem onstrated the formation of indole from tryptophan by Bacterium coli, numerous investigators have attem pted to find th e m echanism of th is reaction (2-5). At least three mechanisms have been suggested. W oods (3) postulated an oxidative degradation of trypto­ phan to yield indole, carbon dioxide, water, and ammonia, 5 atom s of oxygen being used in the process. Baker and H appold ( 6 ) suggested a primary fission into indole and alanine, followed by the oxidation of alanine. Krebs et al. (7) proposed th at the mechanism involved a preliminary oxida­ tion of th e indole ring, followed b y oxidation of the side chain to yield o-am inophenylacetaldehyde, which condensed to indole spontaneously. The data, however, did not substantiate this view and Krebs was led to state that, while Escherichia coli would form indole from o-amino-jS-phenylethanol, via the analogous aldehyde, the mechanism of tryptophanase ac­ tion very probably did n ot involve this compound as an interm ediate. W oods (3) and Baker and H appold (6 ) studied a series of possible oxidative interm ediates between tryptophan and indole, and concluded that an unaltered alanine side chain was necessary for tryptophanase action. M ore recently, D aw son (8 ) found that mepacrine (atabrine) inhibits tryptophanase, and D avres, Daw son, and H appold (9) have been able to recover alanine concurrently with indole formation in the presence of mepacrine. T hey have thus strengthened Baker and H appold’s postu­ late of primary fission to indole and alanine. In the present stu dy, Escherichia coli cells with a very active trypto­ phanase system have been obtained b y growing the culture with aeration. T he cells have been vacuum - or acetone-dried to yield cell preparations which contain m ost of the activity present in the living cells. The enzyme is stable in these preparations and m ay be obtained in a cell-free state by autolysis. The resolution and purification of the enzyme have been accomplished b y precipitation of the cell-free extracts w ith ammonium sulfate and by calcium phosphate adsorption. Pyridoxal phosphate will reactivate the resolved enzym e, thereby adding tryptophanase to the group of vitam in B 6 enzym es. * N u tr itio n F o u n d a tio n F e llo w . 313

314

F U N C T IO N O F P Y R ID O X A L PH O SP H A T E

The produets of th e action of these purified preparations, as well as of the dried cell preparations, are indole, p y ru v ate, and am m onia, in an equim olar ratio. The purified try p tophanase enzym e does not deam inate alanine or serine; th u s neither of these is an interm ediate in the try p to p h a n ase re­ action. Methods Culture— The Crookes strain of Escherichia coli from th e departm ental collection was used. W ith this culture a very active tryptophanase enzym e was obtained by grow th in a m edium composed of 1 per cent tryptone, 1 per cent yeast extract, 0.5 per cent K 2H P 0 4, and 0.1 per cent glucose. The m edium was dispensed in 200 ml. am ounts in 500 ml. E rlenm eyer flasks, inoculated, incubated 4 to (i hours a t 30°, th en placed in a m echanical shaker (approxim ately a hundred 3 inch strokes per m inute) and incubated for an additional 18 to 20 hours. T he cells were harvested with a Sharpies centrifuge. The cell paste from 0 liters of m edium was washed with 250 ml. of w ater, centrifuged, resuspended in 15 to 20 ml. of distilled water, an d dried in vacuo over drierite to yield ab o u t 10 gm. of dried cells. Acetone-dried cells were prepared by p ipetting the washed cell suspensions into 10 volum es of ice-cold acetone. T he cells were collected on a Buchner funnel and washed w ith ether. B oth the vacuumdried and acetone-dried preparations contained tw o-thirds or more of the try ptophanase activ ity present in the living cells, the enzym e being stable in th e dried state. Tryptophanase Delermination— T ryptophanase activ ity was determ ined by m easuring indole form ation. The usual assay was perform ed in 2 ml. volume containing the following: 0.2 ml. of 1 m phosphate buffer, pH 8.3 ; 20 7 of barium pyridoxal phosphate; 50 to 500 y of the cell preparations described above; 2 mg. of L-trvptophan. The enzym e, buffer, anti coenzym e were incubated at, 37° in a volume of 1.8 ml. for 10 m inutes. The substrate (0.2 ml.) was added, the reaction allowed to proceed 10 m inutes, and th en stopped with 0.2 ml. of 100 per cent trichloroacetic acid. The indole was extracted by shaking with 2 ml. of toluene, an d a portion of the toluene layer was removed for analysis. Analytical Methods— Indole was determ ined by E rhlich’s m ethod, modified as follows: From 0.2 to 1 ml. of toluene layer, depending on the level ot indole expected, was pipetted into a colorim eter tu b e and 1 ml. of 5 pei- cent p-dim ethylaininobenzaldehyde in ethyl alcohol was added. The tubes were filled to tin- 10 ml. line w ith an acid alcohol solution (1 liter ot ethyl alcohol plus 80 ml. ot concentrated sulfuric acid), allowed to stan d 10 m inutes, and read in an E velyn colorim eter, w ith th e No. 540

W O O D , G U N S A L U S , A N D U M B R E IT

315

filter. W ith this m ethod, indole can be determined over a range of 1 to 15 t ; the m ethod m ay be used to 2 0 y w ithout great deflection from linearity. Pyruvate— For m ost determ inations, the direct m ethod of Friedemann and H augen (10) w ith 2,4-dinitrophenylhydrazine was used. The iden­ tity of the pyruvate was established b y th e toluene extraction m ethod, the quantitative data agreeing w ith the direct m ethod. A m m onia— Am m onia was determined b y nesslerization after distillation from a Pregl still (11). The reaction m ixture was deproteinized w ith trichloroacetic acid, neutralized to nearly pH 7, and pipetted into the still. 2 ml. of a borate buffer ( 1 2 ) were added and the sample steam -distilled for 5 m inutes, about 6 ml. of distillate being collected. The distillate was nesslerized b y adding 2 ml. of Johnson’s reagent (13) and 1 ml. of 6 n alkali, and then diluted to 10 ml. After 10 m inutes the color was read in the E velyn colorimeter w ith the N o . 490 filter. Results W ith the dried cell preparations, it was found that the quantity of indole formed was proportional to the cell concentration only at the lower levels (Fig. 1). The lack of proportionality of indole production at the higher cell concentrations was later shown to be due to the inhibitory effect of indole, as previously described by Fildes (2). The enzyme could be assayed reasonably well, however, over a range of cell concentrations from 50 to 300 y ; equivalent to 1.5 to 1 2 y of indole formed in a 10 m inute incubation period. As shown in Fig. 2, the rate of indole production* by a given cell concentration also decreased with tim e. Inasmuch as the cell preparations were sufficiently active to give accurate analytical values w ithin a 1 0 m inute period, there was no need for a longer incubation time. The dried preparations did not s&ow an oxygen uptake with trypto­ phan, and so no effort was m ade to run the experiments anaerobically. An attem p t to determine the products of tryptophanase action showed that pyruvate was formed in approximately equimolar ratio to the indole formed. The preparations contained serine and alanine deaminases, which also formed pyruvate; thus one of these m ight be an intermediate in the tryptophanase reaction. The alanine would be considered as a possible intermediate in view of the work of Dawes, Dawson, and Happold (9 ); and serine m ight be considered in view of the formation of trypto­ phan from serine and indole b y the Neurospora enzyme (14, 15). In order to determine whether either of these was an intermediate in the reaction, conditions were sought in which the tryptophanase was active whereas the deaminases were not. In view of the function of pyridoxal phosphate as the coenzyme of tryptophan synthesis with the Neurospora enzym e (15), the possibility of

316

F U N C T IO N O F PY R ID O X A L PH O SPH A T E

its function in th e try p to p h an ase reaction was obvious. A ttem p ts to d em onstrate th e action of pyridoxal phosphate w ith the dried preparations resulted in approxim ately 50 per cent stim ulation of th e ra te of indole form ation. T hus, even in th e dried preparations, th e enzym e system which form s indole from try p to p h a n was p artially resolved an d could be activ ated by pyridoxal phosphate as the coenzyme. 35

14

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F

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TIME IN MINUTES F

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1. T ry p to p h an ase a c tiv ity of dried cell p rep ara tio n . 2 m l. reac tio n volum e containing 0.2 ml. of 1 m phosphate buffer, p H 8.3; 20 y of pyridoxal p h o sp h ate (barium sa lt); dried cells as indicated; w ater to 1.8 m l.; le t s ta n d 10 m in u tes a t 37°; add 0.2 ml. (2 mg.) of i.-try p to p h an ; in c u b ate 10 m inutes a t 37°. F i g . 2. T ry p to p h an ase a c tiv ity and in c u b atio n tim e. C ondition s are th e sam e as in Fig. 1. F

ig

.

Enzyme Purification and Resolution 10 gm. of an acetone-dried preparation were evenly suspended in 500 ml. of water in a Florence flask and placed in a cold room overnight and allowed to freeze. The next m orning the flask was rem oved from the cold room and placed in a 37° w ater bath, where thaw ing an d autolysis were allowed to proceed for 2 hours. The cell debris was rem oved by centrifugation and the su p ern atan t treated a t room tem p eratu re, w ith an equal q u an tity of saturated am m onium sulfate neutralized to a pH of about 8.5. L pon standing in the refrigerator for a short tim e, the precipitate flocculated an d was rem oved by centrifugation. T he su p e rn atan t was satu rated with solid am m onium sulfate and 7 ml. of 0.01 M sodium cyanide

317

W O O D , G U N S A L U S , A N D U M B R E IT

added (final concentration 0.0001 m ) . The solution was allowed to stand in the refrigerator until the precipitate flocculated, and was then centri­ fuged. T he precipitate was suspended in 270 ml. of water and the in­ soluble m atter rem oved b y centrifugation. T he supernatant solution was again treated w ith am m onium sulfate to 55 per cent saturation and the small qu antity of precipitate which formed was discarded. The enzyme was then precipitated by adding amm onium sulfate to 6 8 per cent satura­ tion and th e precipitate removed by centrifugation. This precipitate, which contained about 1 0 per cent of the enzym e originally present in th e cells, was suspended in 50 m l. of water. Pilot experiments were performed to determ ine th e concentration of calcium phosphate gel necessary just to adsorb the enzym e, and the indicated am ount was added with m ixing and allowed to stand 10 minutes. The phosphate gel was T a b le

I

Pyruvate Form ation by Cell P reparation and by Purified E nzym e T h e c o n d itio n s a re th e s a m e a s in F ig . 1. P y ru v a te form ed S u b s tra te 0.5 m g. cell p re p a ra tio n

0.05 m l. cell-free enzym e

y L - T r y p t o p h a n ................................................................................................

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collected by centrifugation and washed with five 250 ml. portions of dis­ tilled water, after which the enzym e was eluted from the gel with 50 ml. of 1 m phosphate buffer, pH 6.0. The enzym e at this stage of pur­ ification was com pletely resolved and free from serine and alanine deaminases (see Table I). Characteristics of Tryptophanase The influence of tryptophan concentration on the reaction rate is shown in Fig. 3. W ith the cell-free enzym e, th e half maximum rate is obtained w ith 35 7 of tryptophan per ml. (M ichaelis constant (16), K = 2.5 X 10~ 5 m ole per liter). W ith the dried cells, a somewhat higher substrate concentration is required, the rate of indole formation dropping sharply below 2 0 0 7 of tryptophan per ml.

318

F U N C T IO N O F PY R ID O X A L PH O SPH A T E

The coenzyme sa tu ratio n curve for try p to p h a n ase is shown in Fig. 4, th e half sa tu ratio n concentration of barium pyridoxal phosphate being 0.9 7 per ml. (V iclm elis constant, K = 2.1 X 10~6 mole per liter). T hus th e dissociation constant of the pyridoxal phosphate-tryptophanase com­ plex approxim ates th a t of the glutam ic-aspartic transam inase,1 K = 1.5 X

10 Z

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150

200

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500

ML.

F i g . 3. S u b strate sa tu ra tio n curve for try p to p h a n a se . as in Fig. 1; 0.03 ml. of purified enzym e.

C ondition s are th e sam e

z s o 2

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S o o

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PYRIOOXAL

PHOSPHATE

PER ML.

4. Coenzyme s a tu ra tio n curve for try p to p h an ase. a s in Fig. 1; 0 .0 3 ml. of purified enzym e. F ig .

C onditions a r e th e same

11)“ ';. hut higher th an th a t of the tyrosine decarboxylase,2 K = 1.5 X 10~8 mole per liter. B oth th e tryptophanase and th e transam inase reactions are run at neutral reaction, b ut the tyrosine decarboxylase is run a t an acid pH. 1 O ’K ane, D . L.. and G unsalus, I. ./. lUol. Chem., in press. 2 G unsalus. I. C., and U m breit, W. W., unpublished d ata.

W O O D , G U N S A L U S , A N D U M B K E IT

319

Z

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- PYRUVATE •©-AMMONIA------

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F ig . 5 . I n f lu e n c e o f p r o d u c t s u p o n , t r y p t o p h a n a s e a c t i v i t y . C o n d itio n s a re th e s a m e a s i n F i g . 1 . 0 .0 3 m l . o f p u r i f i e d e n z y m e ; in d o l e p y r u v a t e a n d a m m o n i a , a d d e d in th e q u a n titie s in d ic a te d , b e fo re th e e n z y m e is a d d e d .

II

T a b le

Inhibition of Tryptophanase by N a C N C o n d i t i o n s a r e t h e s a m e a s i n F i g . 1 ; 0 .0 3 m l. o f p u r i f i e d e n z y m e ; c y a n i d e a d d e d a fte r e n z y m e a n d c o e n z y m e . C o n cen tratio n of cy a n id e

In d o le form ed

M

T

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0 .0 1 .0 5 .2 1 1 .5 1 7 .5 1 7 .5 T a b le

P e r c e n t in h ib itio n

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m

III

Products of Tryptophanase Reaction v o l u m e ; 0 . 7 m l . o f 1 m p h o s p h a t e b u f f e r , p H 8.3; s a l t ) ; 0 .3 m l. o f p u r i f i e d e n z y m e . W a t e r t o 6 .3 m l . ; l e t s t a n d 1 0 m i n u t e s , 3 7 ° ; 0 .7 m l . (7 m g .) o f L - t r y p t o p h a n a d d e d a n d i n ­ c u b a te d 10 m in u te s . C o n d itio n s : 7 m l., r e a c tio n

70 y o f p y r i d o x a l p h o s p h a t e ( b a r i u m

M icrom oles of p ro d u c ts form ed

2057 2101 2102 2103 2103a M o la r r a tio

(a v e ra g e )

In d o le

P y ru v a te

A m m onia

0 .9 2 1 .3 7 1 .4 4 1 .0 2 0 .8 2

0 .9 9 1 .0 9 1 .3 3 1 .0 7 1 .2 5

0 .1 3 1 .1 2 1 .3 4 1 .0 3 1 .2 8

1 .0 0

1 .0 5

1 .0 5

320

FUNCTIO N' O F PY R ID O X A L P H O SPH A T E

T ry p to p h an ase is sensitive to indole accum ulation, as indicated by the cell preparations, Figs. 1 an d 2, and as shown with the cell-free enzyme, Fig. 5. The presence of p y ru v ate plus am m onia has a slight influence. The enzym e is sensitive to cyanide (Table II). This sensitivity, how­ ever, m ay not indicate an iron catalyst but a reaction betw een cyanide and th e free carbonyl group of pyridoxal phosphate. A sim ilar sensitivity of pyridoxal phosphate-containing enzymes to cyanide is m entioned by Blaschko (17) and by fad e (18) for the anim al and bacterial decarboxylases respectively. Products of Tryptophanase Reaction The try p to p h an ase reaction, as catalyzed by the dried cell preparations and by the partially purified enzyme, can be expressed b y the following equation: try p to p h a n —> indole + pyruvic acid + am m onia. Analyses of the products form ed in several experim ents with the purified enzyme are shown in Table III. DISCUSSION

From the d ata presented, it is apparent th a t the try p to p h an ase system of Escherichia coli is n o t the reversal of the indole and serine condensation which leads to try p to p h a n with the Xcurospora enzyme. W ith purified try p to p h an ase the products are indole, pyru v ate, and am m onia. Serine and alanine do not yield p yruvate, and are not, as such, interm ediates in th e reaction. The interm ediates postulated by various investigators (3, 5, 7), with tho possible exception of am ino acrylic acid (12), do not appear to be involved. SUMMARY

T ryptophanase has been obtained in a cell-free sta te from Escherichia coli and has been p artially purified. The enzyme has been resolved and shown to require pyridoxal phosphate as the coenzyme. I he enzym e preparation catalyzes the breakdow n of try p to p h a n ac­ cording to the following reaction: try p to p h a n —> indole + pyruvic acid + am m onia. No oxidation occurs in the process, nor floes alanine or serine occur as an interm ediate. B IB L IO G R A P H Y

1. 2. 3. 4. 5.

H opkins, F. G., and Cole, S. W., J . Physiol., 29, 451 (1903). F ildes, P ., Biochem. J . , 32, 1600 (1928). Woods, D . D ., Biochem. J ., 29, 640 (1935). H oods, D . D ., Biochem. J ., 29, 649 (1935). H appold, F . C., and H oyle, L., Biochem. J . , 29, 1918 (1935).

W O O D , G U N S A L U S , A N D U M B R E IT

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

321

B a k e r , J . W . , a n d H a p p o l d , F . C . , Biochem . J ., 3 4 , 6 5 7 ( 1 9 4 0 ) . K r e b s , H . A . , H a f e z , M . M . , a n d E g g l e s t o n , L . V . , Biochem. J ., 3 6 , 3 0 6 ( 1 9 4 2 ) . D a w s o n , J . , Biochem. J ., 4 0 , p . x l i ( 1 9 4 6 ) . D a w e s , E . A . , D a w s o n , J . , a n d H a p p o l d , F . C . , Biochem. J ., 4 0 , p . x l v ( 1 9 4 6 ) . F r i e d e m a n n , T . E . , a n d H a u g e n , G . E . , J . B iol. Chem., 1 4 7 , 4 1 5 ( 1 9 4 3 ) . N ie d e r l J . B ., a n d N i e d e r l , V ., M i e r o m e t h o d s o f q u a n t i t a t i v e o r g a n ic a n a l y s i s , N e w Y o r k , 2 n d e d itio n (1 9 4 2 ). C h a r g a f f , E . , a n d S p r i n s o n , D . B . , J . B iol. Chem., 1 5 1 , 2 7 3 ( 1 9 4 3 ) . J o h n s o n , M . J . , J . B iol. Chem., 1 3 7 , 5 7 5 ( 1 9 4 1 ) . T a t u m , E . L . , a n d B o n n e r , D . , Proc. N a t. A cad. Sc., 3 0 , 3 0 ( 1 9 4 4 ) . U m b r e i t , W . W . , W o o d , W . A . , a n d G u n s a l u s , I . C . , J . B iol. Chem., 1 6 5 , 7 3 1 ( 1 9 4 6 ) . L i n e w e a v e r , H . , a n d B u r k , D . , J . A m . Chem. Soc., 6 6 , 6 5 8 ( 1 9 3 4 ) . B la s c h k o , H ., in N o r d , F . F ., a n d W e rk m a n , C . H ., A d v a n c e s in e n z y m o jp g y a n d r e la te d s u b je c ts , N e w Y o r k , 5 , 6 7 (1 9 4 5 ). G a le , E . F ., in N o r d , F . F ., a n d W e rk m a n , C . H ., A d v a n c e s in e n z y m o lo g y a n d r e la te d s u b je c ts , N e w Y o rk , 6 , 1 (1 9 4 6 ).

R e p r in te d fro m T h e J o u r n a l o p B io l o g ic a l C h e m is t r y V ol. 181, N o . 1, N o v e m b e r, 1649

S E R IN E A N D T H R E O N IN E D E A M IN A S E S OF E SC H E R IC H IA COLI: A C TIV A TO R S FO R A C E L L -F R E E EN ZY M E* By

W . A . W O O D

and

I. C . G U N SA L U S

(From the Laboratory o f B acteriology, Ind ia n a U n iversity, Bloomington) (R e c e iv e d f o r p u b lic a tio n , J u n e 1 7 , 194 9 )

Serine has been reported by m any workers to be deaminated by a variety of bacterial cells and tissues, but this process was first studied in detail by Gale and Stephenson in 1938 (1 ). These workers followed the serine deaminase of Escherichia coli by measuring the release of ammonia by resting cell suspensions. The reaction proceeded anaerobically, thereby distinguishing it from the oxidative deaminases. Aging of cell suspen­ sions caused a loss of deaminase activity, which could be prevented by the addition of reducing agents, such as glutathione or formate, or by adenylic acid. Chargaff and Sprinson (2 , 3) studied serine and threonine deaminases, using toluene-treated suspensions of E. coli and found that pyruvate and a-ketobutyrate, respectively, accumulated as the products of anaerobic deam ination. N either the O-ethers of serine nor phosphoserine were deam inated anaerobically. On the basis of these findings, Chargaff and Sprinson suggested desaturation as the mechanism. Binkley (4) obtained cell-free extracts of serine deaminase from E. coli which were inactivated by dialysis. The activity was restored by the addition of zinc ions. Lichstein et al. (5, 6 ) in studying the m etabolic r61e of biotin inactivated the serine and threonine deaminases of E. coli by aging cell suspensions in phosphate buffer at pH 4. The activity was restored by the addition of biotin or adenylic acid. W ith a cell-free preparation, only yeast extract caused partial reactivation. From this evidence it was sug­ gested th at a coenzym e form of biotin is present in yeast extract (7). In the present stu d y, active serine and threonine deaminases have been obtained by growing E . coli in deep medium w ithout carbohydrate. Vac­ uum-dried cells were prepared which contained m ost of the deaminase ac­ tiv ity present in the living cells, and which differed from the living cells only in the requirem ent of adenylic acid for activation. The deaminases were freed from th e cells by autolysis, and purified b y ammonium sulfate precipitation and adsorption on calcium phosphate gel. The purified enzym e required both adenylic acid (AM P) and glutathione (GSH) for a c tivity. T he enzym e, as prepared, deam inated both serine and threo­ nine. D uring serine deam ination, sim ultaneous inactivation toward both substrates occurred. * T h i s w o r k w a s s u p p o r t e d in p a r t b y t h e O ffic e o f N a v a l R e s e a r c h . 171

' ! H IM . A N D T H R E O N IN E D E A M IN A SE S

M clhods C u l t u r e - T he Crookes strain of E . coli, em ployed previously for studies

of th e arginine and glutam ic acid decarboxylases (8) and of try p to p h a n ase (9), w as used. F o r active deam inase production, th e cells were grown w ith o u t aeratio n in a m edium com posed of 2 per cent try p to n e , 1 per cent yeast ex tract, and 0.5 per cent dipotassium phosphate. To obtain a large crop of cells, 10 liter batches were grown in 2 \ gallon reagent bottles. The m edium was in oculated w ith 3 per cent of a 6 to 9 hour culture and in cubated 12 to 14 hours a t 37° (final pH 6.8 to 7.2). T he cells were harv ested w ith a Sharpies centrifuge, the cell p aste resuspended in 0 . 1 m phosphate buffer, pH 7.8, containing 3 X 10~3 m glu tath io n e, and dried in vacuo over D rierite (yield, 3.5 gm . of dry cells per 10 liters of m edium ). The deam inase a c tiv ity of the dried cells was ap proxim ately 560 gl. of p y ru v ate per mg. of d ry w eight per hour w ith L-serine and about 890 gl. of a-k e to b u ty ra te w ith L-threonine. Determination of Serine and Threonine Deaminase— T he deam ination of serine an d threonine has been shown to yield equim olar am ounts of am m o­ nia and p y ru v ate or a -k eto b u ty rate (3). Since th e vacuum -dried cells did not m etabolize these keto acids, th e deam inase a c tiv ity could be fol­ lowed by m easuring the rate of keto acid form ation. T he enzym e activ ity was assayed in a 1 ml. volum e containing the following: 0.1 ml. of 1 m phosphate buffer, pH 7.8, 0.1 m l . of 7 X 10-3 m adenylic acid, w ater to 0.89 ml., 0.01 ml. of enzym e and 0.1 ml. (1 mg.) of L-serine or L-thrconine. Before addition of the enzym e and su b strate, the assay tubes were brought to 37°. A fter su b stra te addition, the reaction was allowed to proceed for 10 m inutes, th en stopped w ith 0.5 ml. of 20 per cent trichloroacetic acid, the protein rem oved by centrifugation, and a 1 ml. portion of the superna­ tan t rem oved for analysis. A unit of serine or threonine deam inase was a rb itra rily defined as the am ount of enzym e necessary to form 1 jim of p y ru v ate or a-k eto b u ty rate in 10 m inutes under the above experim ental conditions. Pyruvate— F or most determ inations, the direct m ethod of Friedem ann and H augen (10) was used. Analyses by the ex tractio n procedure of hriedem ann and H augen agreed w ith th e results of the direct m ethod. a - K e to b u ly r a te :- a -K eto b u ty ra te was also determ ined by the direct m ethod oi Friedem ann and H augen. T he color was com pared w ith a standard curve prepared from crystalline a-k eto b u ty rate-2 ,4 -d in itro p h en y lh y d razone, m .p. 204-205° (uncorrected). T he color, when read in the E velyn colorim eter with a 515 mg filter, was linear up to 70 y of a -k e to b u ty ra te . Results The enzym e assay w as standardized by using graded am ou n ts of dried cells. As is show n in Fig. 1, DL-threonine w as d eam in ated m ore rapidly

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A . WOOD

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than L-serine, the l’ate being proportional to cell concentration with each substrate. T he form ation of 20 to 120 y of pyruvate or 35 to 230 y of a-ketobutyrate thus corresponds to 50 to 300 y of dry weight of cells. Since oxygen did not affect enzym e activity, assays were run aerobically. L-Threonine w as used in later experim ents w ith the purified enzyme and found to be deam inated more rapidly than the d l m ixture, thus indicating possible inhibition by the d isomer. SER IN E- TH REO N IN E D E AM INASE - E.CO LI , 3 .0

?ol - T H R E O N I N E

in 2 /

0

100 juG

200

D R IE D

300

400

CELLS

PER

500

M L.

F i g . 1 . S e r in e an d th r e o n in e d ea m in a se a c t iv it y o f va cu u m -d r ied c e lls . C o n d i­ t io n s , 0.1 m l. o f m p h o s p h a te b u ffer, p H 7 .8 ; 0.1 m l. (2.5 m g .) o f a d en o sin e-5 -p h o sp h a te ; 0.6 m l. o f w a ter; a llo w e d to s ta n d 5 m in u te s a t 37°; c e lls as in d ic a te d a n d w a ter to 0 .9 m l.; 0.1 m l. (1 m g .) o f L -serine or (2 m g .) D L -threon in e; in c u b a te 10 m in n u te s a t 37°; ad d 0 .5 m l. o f 20 per c e n t tr ic h lo r o a c e tic a cid . W e w ish to th a n k D r . H . E . C a rter fo r k in d ly fu rn ish in g th e L-serine.

In the a ctivity, adenylic enzym es

dried preparations, both deaminases required adenylic acid for in contrast to the resting cells. In order to study the role of acid in the absence of interfering reactions, extraction of the and partial purification were undertaken. Cell-Free Enzyme

gm. of vacuum -dried cells were suspended in 2 0 0 ml. of glutathionephospbate buffer and the deaminases extracted by freezing, thawing, and autolysis. The cell-free extracts, obtained by centrifugation of the autol­ ysate, contained about 50 per cent of the total activity. The enzymes 4

17-1

■E R IX E A N D T H R E O N IX E

D E A M IN A SE S

were precip itated by 40 per cent sa tu ratio n w ith am m onium sulfate, a d ­ sorbed on calcium p h osphate gel, and eluted w ith phosphate buffer. T he details of the purification are shown in the flow sheet. T h e first eluate Flow Sheet for Purification of Serine and Threonine Deaminases Suspend 1 gm. v acuum -dried cells (4100 u n its serine deam inase, 4570 u n its th reo n in e deam inase) in 50 ml. 0.1 M p h o sp h ate buffer, p H 7.8, co n tain in g 6 X 1 0 " 3 m GSH. Freeze and thaw twice, au to ly ze 4 to 5 hrs. a t 37°; centrifuge

D iscard

1870 u n its serine deam inase, 2420 un its th re o n in e d eam inase Add (NHdaSCh to 40% s a tu ra tio n ; centrifuge

R edissolve in 5 ml. 0.1 m phosphate buffer, pH 7.8, co n tain in g 3 X 1 0 ^3 m adenylic acid; ce ntrifuge

D iscard

1450 u n its serin e deam inase, 1020 u n its th re o n in e deam inase A dd 50 ml. 1:10 d ilu tio n of C ajfP O d ’ gel, stir, centrifuge

W ash w ith four 12 ml. portions distilled w ater

F lu te w ith 5 ml. buffer, pH 7.8

D iscard

m

phosphate

D iscard

D iscard washings

503 units serin e deam inase, 525 units th reonine deam inase; 10 to 15% recovery Add adenylic acid to 3 X 10 3 m

from the calcium phosphate gel contained 10 to 15 per cent of the activ ity present in th e dried cells. W hen stored in the frozen sta te , the enzymes were stable for several m onths; however, when stored a t 0 ° w ithout aden­ ylic acid, in activ atio n occurred. T he enzym e units recovered a t each step

W . A . W O OD A N D I .

175

C. G U N S A L U S

in the purification and th e degree of resolution w ith I’espect to adenylic acid (and glutathione) are shown in Table I. The degree of resolution was obtained b y expressing the increase in rate of deam ination due to addition of th e activator as per cent of the maxim um rate (activator present). The relative serine and threonine deaminase a ctivity at each step in the purification is shown in the last column of Table I. T he purity index was expressed as th e ratio of threonine deam inase units to protein content, as determ ined by the biuret test of R obinson and H ogden ( 1 1 ). Characteristics of Serine and Threonine Deaminases T he purified extract contained both serine and threonine deaminases in virtu ally th e same proportions as were present in dried cells (Table I). This suggested a sim ilarity of properties, if not the id en tity of the enzym es. T a b le

I

P u rification of Serine and Threonine Deam inases R esolution S te p N o.

1 . C e ll s u s p e n s io n (in G S H - 0 . 1 m p h o s ­ p h a te , p H 7 .8 ) 2 . C e ll- f r e e e n z y m e (fro m c e lls fro z e n , a u to ly z e d a t 3 7 °, 5 h r s .) 3 . P p t . fro m 4 0 % s a tu r a te d (N H d jS C h 4 . E l u a t e f r o m C a a ( P O i )2 g e l ( i n 1 m p h o s p h a te , p H 7 .8 )

A c tiv ity AM P

GSH

u n its

per cent

per cent

per cent

4100

100

82

32

1870

46

1450 593

35 15

L-Serine DL-Threonine

0 .9 0 0 .7 7

94

99

1 .4 2 1 .1 3

For com parative purposes, the enzym e characteristics were investigated by use of both serine and threonine as substrates. T he influence of serine and threonine concentration upon enzym e activ­ ity is shown in F ig. 2. The half maximum activity was obtained w ith approxim ately 305 y per ml. of L-serine or L-threonine. This corresponds to a M ichaelis constant (12) for substrate-enzym e of 3.5 X 10~ 3 and 3.0 X 10 - 3 mole per liter, respectively. T he presence of the d isomer of serine or threonine depressed deam ination by about 50 per cent. Cysteine, which differs structurally from serine only in the polar group on the /3-carbon, has been shown by Desnuelle and From ageot (13) to be deaminated by E. coli in a manner similar to serine, the products being pyruvate, ammonia, and hydrogen sulfide. In the light of these similarities, desulfurase activ­ ity as determ ined by pyruvate form ation was measured, but no activity was found. B inkley (4) has suggested that enolase, in addition to converting

17(5

S E R IN E

AND T H R K O K IN E D E A M IN A SE S

2-phosphoglyeeric acid to phosphopyruvate, also catalyzes the deam ina­ tio n of cysteine and serine w ith th e form ation of pyru v ate. Adenylic A cid— As with the dried cells, both serine and threonine deam ­ inases of th e purified extract were activ ated by adenylic acid. Adenosine5-phosphate obtained by hydrolysis of adenosine trip h o sp h ate (A TP) or prepared by the yeast ferm entation of adenosine1 gave the sam e activation (Table II). O ther nucleosides and nucleotides including adenosine, adenosine-3-phosphate, and A T P were ineffective, th u s indicating th at adenosine-o-phosphate is specifically required. As is shown in T able II, y east

Z

5

SUBSTRATE PURIFIED

SATURATION

DEAMINASES I

o

z

oUJ 2 tv o

li_

-= -< » l-SERINE —

o 0.6 V-

L £J

1.0 20 MILLIGRAMS SU BSTR A TE

PER

5.0 ML

Fiti. 2. .S ubstrate sa tu ra tio n curves for serin e am i th re o n in e deam in ases. C o n ­ d itions as in Fig. 1 except as follows: 0.1 nil. of l.li X H C m g lu ta th io n e ad ded w ith a denosine-o-phospliate: allowed to sta n d 10 m inutes at 117° w ith enzym e; su b stra te levels as indicated.

extract, which was found bv bichsteiu (7) to activ ate these deam inases in the absence of adenylic aeitl or biotin, was ineffective with the purified enzymes. The adenylic acid activation curves for bolh deam inases are shown in Fig. 3. T he concentration necessary for half m axim um activation is about 400 7 per ml. with serine and about 245 y per ml. with threonine. These correspond to Michaelis constants of 1 X 10~:l and 0.7 X 10' 3 mole per liter respectively. ’ We wish to th a n k the H nist Biseholl C om pany, Iv o ry to n , C o n n ec tic u t, for a su pply of adenosine-5-phosphate.

177

W . A . W OOD A N D I . C. G tlN S A L U S '

Glutathione For activity, the purified deaminase required, glutathione in addition to adenylic acid (Table II I). Reducing agents, including T a b le

II

A ctiva tio n of Serine and Threonine D eam inases by A denosine-5-Phosphale K e to acid form ed s K e to b u ty ra te

N o n e ..................................................................................... A d e n o s i n e .......................................................................... A d e n o s i n e - 3 - p h o s p h a t e ........................................... A d e n o s i n e - 5 - p h o s p h a t e ( f r o m A T P ) .................. A d e n o s in e -5 -p h o s p h a te (y e a s t fe rm e n ta tio n ) A d e n o s in e t r ip h o s p h a t e ............................................ G u a n y l i e a c i d ................................................................. Y e a s t e x t r a c t ( 1 m g . p e r m l . ) ............................... A d d itio n s 7 X

T

r

0 0 0

0 0 0

53

120

49

123

3 .3 0

4. 0 0

0

IO -3 m e x c e p t a s i n d i c a t e d .

2 .4 AD EN OSINE- 5 - PH OSPH ATE PU R IFIE D

o

D EA M IN A SE

SATURATION i

I

l -TH R EON IN E

Qi u 2

DC

o u.

Q U < o

0.8

^

l -SERIN E

I-

x: 2

I lJ

M IL LIG R A M S

A D E N O S IN E -5 - PHOSPHATE

PER

ML.

A d e n o s in e -5 -p h o s p h a te s a tu r a tio n c u rv e fo r s e rin e a n d th re o n in e d e a m i­ n a s e s . C o n d i t i o n s a s i n F i g . 2 e x c e p t 0 .1 m l . (1 m g . ) o f L - t h r e o n i n e a n d a d e n y l i c a c i d le v e ls a s in d ic a te d . W e a r e in d e b te d to D r . E . E . H o w e o f M e rc k a n d C o m p a n y , I n c ., R a h w a y , N e w J e rs e y , fo r a s a m p le o f L -th re o n in e . F ig . 3 .

cysteine, sodium thioglycolate, and ascorbic acid, were ineffective. H ow ­ ever, sodium sulfide and sodium cyanide did cause partial reactivation. Since glutathione forms complexes with heavy m etals and acts as a reduc-

ITS

S E R IX E AX'D T H R E O X IX E D E A M IX A SE ?

ing agent as well, other com plex-form ing agents were tested. B ipyridyl, pyrophosphate, and gum arabic were with-

8-hydroxyquinoline, histidine,

T able

III

Activation of Parlialhj Purified Serine and Threonine Deaminases C onditions as in Fig. 3 except th a t g lu ta th io n e and adenylic acid were ad d ed as in d icated . K eto acid form ed C o n c en tratio n

A dditions

P y ru v a te

m x io-> N one . . AM P . G S H .............. A M P + GSH

6. 8 12.8 6.8, 12.8

2

7 0 .5 2.3 6.1 107

a -K e to b u ty ra te I

1 !

y 0.0 3.6 4.9 134

THREONINE DEAMINASE GLUTATHIONE ACTIVATION

2 2 .4 o

z oUi 5 cr O

AMP PRESENT

La

(>T I3 m o t-

u 5 4

AMP ABSENT

0

5

10

15

20

TIME WITH GSH IN MIN. F ig . 4. A ctivation of th re o n in e deam inase by g lu ta th io n e . C o n d itio n s as in Fig. 1 except as follows: Enzym e in c u b ated w ith glutathione, for th e tim es in d ic a te d ; adenylic acid added before or a fte r in c u b atio n w ith g lu ta th io n e as in d ic a te d ; su b ­ s tr a te , D i.-threonine dl-THREONINE (O R + l -SERINE)

UJ

O Ia:

dl-THREONINE

LlI

l -S E R IN E

— / \ ------------F —

i---------

> - / — dl-THR EO NINE ADDED

10 15 20 25 TIME IN MINUTES

30

tiG . 7. In a c tiv a tio n of th reo n in e deam inase by serine. C onditio n s as in Fig - except for s u b stra te ad d itio n s and in c u b atio n tim es as in d icated .

W . A . W OOD A N D

I. C. G U N S A L U S

181

D eam ination of serine and threonine b y the purified extract m ay be due to the presence of tw o similar enzym es or one enzym e catalyzing the two reactions. A s is shown in T able I, the purified preparation contained the deaminases in about th e sam e proportion as the dried cells; also, as shown in Table III, both deam inases were activated by adenylic acid and gluta­ thione. A ttem p ts were therefoi’e made to show the presence of separate enzym es catalyzin g the two deam inations. The rate of keto acid forma­ tion in the presence of threonine w ith increasing levels of serine is shown in Fig. 6. T he data indicate th at the initial rate of deam ination of a serine-threonine m ixture was interm ediate betw een the rates obtained w ith serine or threonine alone. Since th e rates were not additive, com petition for a single enzym e is suggested. A second fact which suggests the iden­ tity of th e enzym es is the loss of deaminase activ ity for both substrates during incubation w ith the m ixture, the rate being proportional to the serine concentration. T o show th e presence of an independent threonine deaminase, conditions were em ployed in w hich serine deaminase was inactive; i.e., after 10 m in­ utes incubation of the enzym e w ith L-serine (Fig. 5). The results, as recorded in Fig. 7, show th at after incubation w ith serine the enzym e did not deam inate threonine, thereby indicating th at threonine deaminase was incapable of functioning independently of serine deaminase. This suggests the id en tity of th e two enzym es. Com petitive inhibition of threonine deam ination by serine appeared unlikely, since deam ination of a serinethreonine m ixture occurred at approxim ately the same rate as w ith threo­ nine alone (Fig. 7). SU M M A R Y

Serine and threonine deam inases have been obtained from Escherichia coli and partially purified. The enzym e has been resolved and shown to require adenosine-5-phosphate and glutathione for activity. Serine and threonine deam inases occurred in the extracts in approxi­ m ately the sam e ratio as th e dried cells, were activated b y the same con­ centrations of adenylic acid and glutathione, and threonine deamination disappeared when serine deaminase was inactivated. These facts suggest that both substrates m ay be activated by a single enzyme. B IB L IO G R A P H Y

1. 2. 3. 4. 5.

G C C B L

a l e , E . F . , a n d S t e p h e n s o n , M . , Biochem. J ., 3 2 , 3 9 2 ( 1 9 3 8 ) . h a r g a f f , E . , a n d S p r i n s o n , D . B . , J . B iol. Chem., 1 4 8 , 2 4 9 ( 1 9 4 3 ) . h a r g a f f , E . , a n d S p r i n s o n , D . B . , J . B io l. Chem., 1 5 1 , 2 7 3 ( 1 9 4 3 ) . i n k l e y , F . , J . B io l. Chem., 1 6 0 , 2 6 1 ( 1 9 4 3 ) . i c h s t e i n , H . C . , a n d U m b r e i t , W . W . , J . B iol. Chem., 1 7 0 , 4 2 3 ( 1 9 4 7 ) .

182 6. 7. 8. 9. 10. 11. 12. 13.

S E R IN E A X D T H R E O X IX E D E A M IN A SE S

L ich stein , H . C ., and C h ristm a n , J. F ., J . Biol. Chem., 175, 649 (1948). L ich stein , H. C ., J . Biol. Chem., 177, 125 (1949). U m b reit, W. W ., an d G unsalus, I. C ., J . Biol. Chem., 159, 333 (1945). W ood, W. A., G unsalus, I. C ., and U m b reit. W. W ., J . Biol. Chem., 170, 313 (1947). F ried e m an n , T . E ., an d H augen, G. E ., J . Biol. Chem., 147, 415 (1943). R o binson, H . W ., an d H ogden, C. G ., J . Biol. Chem., 135, 707 (1940). L inew eaver, FI., and B urk, D ., J . A m . Chem. Soc., 56, 658 (1934). D esnuelle, P ., and F ro m ag eo t, C ., Enzymologia, 6 , 80 (1939).