Studies on the aerobic oxidatin of fatty acids by bacteria

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STUDIES ON THE AEROBIC OXIDATION OF FATTY ACIDS BY BACTERIA

A Dissertation Presented to the Faculty of the Graduate School The University of Southern California

In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy

by John Harold Silliker September 1950

UMI Number: DP23891

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p^. p, i3i 'si Si-ry T h i s d isse rta tio n , w r i t t e n by

...... .CTQHN..JHAJSQLlD..SJ1LLIKER........ u n d e r the g u id a n c e o f his.... F a c u lt y C o m m itte e on S tudies, a n d a p p r o v e d by a l l its m em bers, has been pre se n te d to a n d a ccep ted by the C o u n c i l on G ra d u a te S tu d y a n d R esearch, in p a r t i a l f u l ­ f i l l m e n t o f re q u ire m e n ts f o r the degree o f DOCTOR

OF

P H IL O S O P F IY

Dean

C om m ittee on Studies

ACKNOWLEDGMENTS The author wishes to express his sincere apprecia­ tion of the personal interest shown by so many persons in his work— His thanks to Drs. M. D. Appleman, James W. Barth­ olomew, Walter Marx, Charles Pait, Robert P. Williams, and to Dean Harry J. Deuel, the members of his committee on studies, for their constructive criticism, for their gener­ ous advice. His thanks to fellow students Mr. Daniel Ivler and Mr. Eugene P. Hess for drafting and photographing the figures and to Mr. Tod Mittwer whose helpful suggestions clarified difficulties both in research and in the draft­ ing of this manuscript. Especial and deepest appreciation to Dr. S. C. Rittenberg for his unstinted interest and encouragement, but most of all, for the quality of his guidance.

TABLE OP CONTENTS CHAPTER I.

PAGE

INTRODUCTION ................................

1

Stimulatory effects produced by fatty a c i d s ...............................

2

Fatty acids as oxidizable substrates for microorganisms .......................

5

Mechanisms of fatty acid oxidation in a n i m a l s .............................

14

Simultaneous adaptation— a possible means of studying the mechanism of fatty acid oxidation of microorganisms II.

..........

20

THE NATURE OF THE ENZYMES, CONSTITUTIVE OR ADAPTIVE, IN FATTY ACID OXIDATION BY VARIOUS BACTERIA:

III.

A S U R V E Y .....................

26

Experimental........... .................

27

Discussion...............................

37

S u m m a r y .................................

38

APPLICATION OF THE TECHNIQUE OFSIMULTANEOUS ADAPTATION TO THE STUDY OF FATTY ACID OXIDA­ TION IN SERRATIA M A R C E S C E N S ..............

39

Experimental

39

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

.

Oxidative patterns in relation to growth on capric acid m e d i u m ................

39

iii CHAPTER

• PAGE Oxidative patterns in relation to specific adaptation to various acids . .

A3

The response of Escherichia coli #2 to growth on and exposure to

fatty acids

.

49

Permeability as a factor in the lag periods

57

The possibility of a single

59

Discussion......................

35

enzyme . . . .

The possibility of the accumulation of an intermediate . .'............

63

Distinct reaction chains merging in a com­ mon intermediate ..................... S u m m a r y ........................ IV.

64

67

THE EFFECT OF CELL POISONS ON FATTY ACID OXIDATION BY S E R R A T I A ..........

68

Experimental....................

70

Effect of DNP and sodium azide on oxida­ tion at pH 7 * 0 ..............

12

The oxidation of fatty acids at pH 8.0 in the presence and absence of DNP

....

Evidence that pelargonic and capric acids are completely oxidized at pH 7*0 in the presence of D N P .............

89

Discussion......................

90

S u m m a r y ........................

97

85

iv CHAPTER V.

PAGE

STUDIES ON THE RELATIONSHIP OP VARIOUS PATTY ACID DERIVATIVES TO THE PRIMARY OXIDATIVE REACTIONS IN FATTY ACID O X I D A T I O N ........ Experimental . . ....................... .

99

100

The oxidation of caprate derivatives by glucose and caprate-grown cells

....

101

The relationship between adaptation to caprate derivatives and the oxidation of other d e r i v a t i v e s ...................

105

The effect of adaptation to caprate deriva­ tives on the oxidation of capric, caprylic, and undecylic a c i d s ..............

VI.

10 7

Discussion...................

108

S u m m a r y .................................

118

THE RELATIONSHIP BETWEEN THE CITRIC ACID CYCLE AND THE OXIDATION OF FATTY ACIDS BY SERRATIA MARCESCENS..............

120

Experimental........; ...................

121

The oxidation of citric acid cycle com­ pounds by Serratia....................

122

The .effect of prior oxidation of succinate and malate on the oxidation of capric a c i d .................................

12 2

V

CHAPTER

PAGE The effect of cooxidation of citric acid cyclecompounds.....................

VII.

12 3

Discussion.................................

129

S u m m a r y ...................................

130

ATTEMPTS TO OBTAIN ENZYMATICALLY ACTIVE PRE­ PARATIONS PROMS E R R A T I A .....................

132

Experimental...............................

134

Dry cell p r e p a r a t i o n s ....................

134

Acetone dried preparations ................

137

Freezing and thaw i n g......................

138

A u t o l y s i s ...............................

139

Discussion.................................

139

S u m m a r y ............................ VIII. BIBLIOGRAPHY

142

R E S U M E ..................................

143

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

151

LIST OP TABLES TABLE I.

PAGE Oxidation of Capric and Pelargonie Acids by Various Bacteria . . .

II.

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

30

Lag Periods in the Oxidation of Patty Acids in Relation to Specific Adaptation of Serratia (Alphin) Cells to Various Acids

III.

.

44

Effect of 2:4-Dinitrophenol on the Oxidation of Fatty Acids by Serratia Marcescens ( A l p h i n ) ............................

IV.

74

The Effect of pH on the Oxidation of Patty Acids by Serratia (Alphin) Cells in the Presence and Absence of 2:4-DInitrophenol .

V.

8l

Lag Periods In the Oxidation of Capric, Undecylic, and Caprylic Acids in Relation to Adaptation to Capric Acid Derivatives . . .

VI.

Capric Acid Oxidation in Relation to the Simultaneous Oxidation of Malate

VII.

109

........

123

Capric Acid Oxidation in Relation to the Simultaneous Oxidation of Oxalacetate, Succinate, and Citrate by Unadapted Cells .

126

LIST OF FIGURES FIGURE 1.

PAGE

Oxidation of Various Fatty Acids by GlucoseGrown Serratia (Alphin) ...................

2.

Oxidation of Various Fatty Acids by GlucoseGrown Serratia ( B a k e r ) ...........

3.

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

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

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

103

Oxidation of Beta keto Capric Acid by Serratia (Alphin)

12.

102

Oxidation of Beta Hydroxy Capric Acid by Serratia (Alphin) .........................

11.

73

Oxidation of Alpha-Beta Unsaturated Capric Acid by Serratia (Alphin)..................

10.

50

Oxidation of Fatty Acids by Adapted and Unadapt­ ed Cells in the Presence and Absence of DNP .

9.

47

Oxidation of Caprylic and Caproic Acids by E. Coli # 2 ...............................

8.

42

Oxidation of Capric Acid by Serratia (Alphin) Cells Specifically Adapted to Other Acids . .

7*

4l

Oxidation of Various Fatty Acids by CaprateGrown Serratia (Baker)

6.

36

Oxidation of Various Fatty Acids by CaprateGrown Serratia (Alphin)

5»

35

Oxidation of Capric Acid by Four Strains of Pseudomonas SP.

4.

33

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

104

The Oxidation of Caprate in the Presence and Absence of M a l a t e .........................

124

CHAPTER I INTRODUCTION The effects of fatty acids on microorganisms are many and diverse, but the influence exerted usually takes one of three forms: 1.

The acids may be toxic, causing inhibition of

growth, cessation of respiration, or death of the cells. 2.

They may be stimulatory, allowing rapid initia­

tion of growth, increased cell yields, and increased res­ piration. 3-

They may serve as oxidizable substrates yield­

ing energy to the cell. A given compound may exert one or more of these influences on a given organism depending upon the experi­ mental conditions imposed.

Thus Bergstrom, Thorell, and

Davide (1946) report inhibition of respiration in the tubercle bacillus by oleic acid in 1:10,000 dilution; Dubos (1949) has repeatedly shown that the same acid stimulates growth of Mycobacterium tuberculosis; Gray (19^9) has re­ cently shown that many species of mycobacteria, including the tuberculosis organism, not only oxidize oleic acid but in fact depend upon it as well as upon certain other fatty acids as a source of energy in the absence of exogenous oxidizable substrates. The toxic action of fatty acids has received

2 considerable attention since such activity suggests possible application of these compounds as therapeutic agents and as a means of killing or inhibiting microorganisms in foods and other commercial products.

Toxicity may be due to hydrogen

ions, to the undissociated molecule, or to the lowered sur­ face tension produced by these surface active materials.

A

detailed discussion of toxicity will not be attempted since two theses from this department will treat this subject ex­ tensively (Simon, 1950; Ward, 1950). Stimulatory effects produced by fatty acids. The most striking study of the stimulatory effects produced by fatty acids is that of Dubos and his co-workers on Myco­ bacterium tuberculosis. This work has recently been re­ viewed in detail (Dubos, 1949).

The tubercle bacillus, in

the common aqueous media used by the bacteriologist, grows in-the form of heavy pellicles or large clumps of organisms which are but little wetted by the water phase of the. medium. This hydrophobic character can be overcome by adding wetting agents to the culture medium.

One class of compounds use­

ful in this respect includes polyoxyethylene derivatives of sorbitan esters of long chain fatty acids, commercially known as Tween compounds.

As a result of adding these sub­

stances to culture media, dispersed and homogeneous cultures of tubercle bacilli can be obtained after one week of incuba­ tion.

Likewise, with proper care being taken to overcome

3 toxic effects, palmitic, stearic, oleic, linoleic, arachidonic, and lignoceric acids (as sodium soaps or water soluble esters) increase yields of avian and human tubercle bacilli from serum albumin medium, and the yields increase in direct proportion to the concentration of fatty acid in the medium. With the use of bacteria as a tool in vitamin assay came the necessity for growing organisms in chemically de­ fined media.

Quite frequently it was found that fatty

acids were a limiting factor for growth in such media. Bauemfeind, Sotier, and Boruff (19^2) found that oleic, stearic, and palmitic acids stimulated the production of lactic acid by Lactobacillus easel in the presence of suboptimal amounts of riboflavin or pantothenic acid, while linolenic acid either stimulated or inhibited, depending upon the amount used.

Strong and Carpenter (1942) noted

that oleic and stearic acids exerted strong stimulatory action in riboflavin assay with L. easei, while palmitic and linoleic acids were strong inhibitors.

Neal and Strong

(194-3) observed both stimulation and inhibition of bacterial response in pantothenate assay, depending upon the level of both the vitamin and the acid used.

Krehl, Strong, and El-

vehjem (1943) obtained increased acid production with added fatty acids when Lactobacillus arablnosus was used for the assay of pantothenic acid.

Kodicek and Worden (194-5) re­

ported that there was an increase in acid production with

4 stearic and palmitic acids and an inhibition with un­ saturated acids in the same assay,

Williams and Fieger

(1 9 ^5 ) found that fatty acids stimulated bacterial response when L. easel and L. arablnosus were used in biotin assay. In a later study (Williams and Fieger, 19^7) it was postulat­ ed that biotin functions as a cell permeability factor and that it could be replaced by the proper lipids.

Guirard,

Snell, and Williams (1946) have shown that sodium acetate greatly stimulates the growth rate of Lactobacillus easel and permits rapid initiation of growth.

This property was

found to be shared by the longer chain length saturated acids beginning with caprylic, myristic displaying highest activity. Hutner (1942) obtained data which indicated that oleic acid is a necessary factor for the growth of Listerella organisms.

Collins, Nelson, and Parmelee (1949) found

that certain strains of lactic acid streptococci failed to grow in a basal medium unless oleic acid was added as an accessory factor.

Quite recently, Lein and Lein (1949) have

obtained a mutant strain of Neurospora which requires oleic, linoleic, or linolenic acid for growth in a minimal medium supporting growth of the parent strain. It is clear therefore that fatty acids may play an essential role in the metabolism of many microorganisms. The media usually used for the cultivation of heterotrophic organisms probably contain sufficient amounts of these

5 compounds to meet the requirements for growth.

Were

it necessary to grow many types of bacteria in purely synthetic media, further requirements for fatty acids might become apparent.

It is evident that in some cases

the role of fatty acids is to modify the physical condi­ tions of the environment (in the case of the tubercle bacillus) while in others fatty acids may actually serve functions analogous to bacterial vitamins (in the case in Neurospora).

It is quite likely that these compounds

are much more important in bacterial nutrition than previously supposed and that future investigations on nutritional requirements will reveal that these sub­ stances are important constituents of the chemical environ­ ment. Fatty acids as oxidizable substrates for micro­ organisms . The study of fatty acid metabolism in micro­ organisms has lagged far behind that of carbohydrate and protein metabolism.

Breusch (1948) has pointed out that

a similar situation exists in animal physiology and at­ tributes it to the following: nl.

Fatty acids and their salts are difficultly

soluble in water;

this fact, together with low diffus­

ibility through the cell walls, has made it difficult ex­ perimentally to bring substrate and enzymes together. Therefore incubation of macerated tissue or tissue sections

6 with fatty acids has produced few results. 2.

The poisonous action on tissue respiration of

saponified and therefore water-soluble fatty acids permits only low substrate concentrations in in vitro incubation experiments and may bring about reactions of no consequence in vivo. 3*

The quantitatively relatively small conversion

capacity of tissue enzymes with respect to fatty acids, as compared to their capacity to act on carbohydrates and proteins. 4.

The lack of suitable microanalytical methods

for proper differentiation, isolation, and identification of small quantities of fatty acids and their metabolites.” For the most part these difficulties are to be ex­ pected in the study of microbial metabolism of fatty acids and explain in part the dearth of information on the mechanisms of attack by microorganisms. Anaerobic transformations of fatty acids by micro­ organisms have been subjected to more detailed investiga­ tion than aerobic conversions.

Probably the most important

of these is the well known reduction of butyric acid to butyl alcohol which occurs in the acetone-butyl alcohol fermentation (Prescott and Dunn, 1940).

Similarly,

propionic acid is reduced to propyl alcohol by Clostridium acetobutylicum (Blanchard and MacDonald, 1935) and by

7 Aerobacter indologenes (Mickelson and Werkman, 1939). Schdnbrunner (1940) has demonstrated the saturation of oleic acid by pure cultures of bacteria, and Rosenfeld (1948) noted that saturation of fatty acids buried in sedimentary materials increased with depth and traced this to the activity of anaerobic bacteria. Interest in anaerobic transformations of fatty acids was stimulated by the postulate that fatty acids might under­ go decarboxylation and yield hydrocarbons with one less carbon atom than the original acid.

Such a process might

point to the origin of hydrocarbons as a result of micro­ biological processes.

Sohngen (1 9 0 6 ) working with mixed

cultures of bacteria, showed that in an otherwise mineral medium formates, acetates, and normal butyrates were quantitatively decomposed to methane and carbon dioxide. Strong indications were obtained that the decomposition of capronates, caprylates, and caprinates followed the same pathway.

Coolhaas (1 9 2 7 ), working with thermophilic

bacteria, established fermentation of formates, acetates, and isobutyrates with the formation of methane.

All other

acids tried, including butyrate, gave inconclusive results. Taylor (1928) mixed fats and oils with sand and covered the mixture with clay, incubating at room temperature. The cultures thus obtained soon produced gas in sufficient quantity to lift the clay from the sand.

The gas consisted

mainly of methane, but Taylor concluded that the fat was

8 hydrolyzed, and the resulting glycerol converted to methane and the fatty acids to the corresponding paraffins-

Thayer

(1 9 3 1 ) reinvestigated the problem, adding marine mud samples to a mineral salts medium containing salts of various fatty acids.

Methane escaped from the fermenting mixture, but

this was the only hydrocarbon detected in the breakdown of fatty acids under these conditions.

Barker (1 9 3 6 ) was the

first to isolate methane producing bacteria in pure culture. Barker, Ruben, and Kamen (19^0) later showed that methane production arose from the reduction of carbon dioxide, the hydrogen required arising from organic substrates, includ­ ing fatty acids.

Buswell and Sollo (19^8) present evidence

which suggests that in the methane fermentation in mixed culture the gas produced arises through some mechanism other than reduction of carbon dioxide, a simple decarboxyla­ tion of acetic acid appearing most likely.

They suggest that

the mechanism found by Barker et al. in Methanosarcina may be limited to pure cultures of that organism.

The evidence

seems to lead to the conclusion that bacteria do not produce hydrocarbons, other than methane, when acting on fatty acids under anaerobic conditions. In the work of Sohngen (1 9 0 6 ) it was shown that hydro­ gen sulfide was one of the first compounds produced in mud samples containing fatty acids.

Later investigation showed

that sulfide arose as a result of the action of sulfate

9 reducing bacteria which used fatty acids as hydrogen donors for the reduction of sulfate.

The reaction involves con­

version of fatty acids to carbon dioxide and hydrogen, with the hydrogen being used to convert a corresponding amount of sulfate to sulfide (Baars, 1930). A unique mode of fatty acid metabolism has been discovered and thoroughly studied by Barker and his co­ workers.

It was shown that Clostridium kluyveri obtains

energy from the synthesis of fatty acids (Barker, 1937)• This anaerobic organism converts two-carbon compounds (ethanol and acetate) to fatty acids of four and six car­ bons (Bomstein and Barker, 1948a, 1948b).

Tracer experi­

ments revealed that this conversion is an oxidationreduction process in which ethanol is oxidized to a twocarbon compound ("active” acetate) that is in approximate equilibrium with acetate.

This active acetate is condensed

with acetate to a four-carbon compound that serves as an oxidant for the reaction and is reduced to butyrate; in a similar manner, the "active” acetate derived from another molecule of ethanol may condense with butyrate to form caproate (Barker, Kamen, and Bornstein, 1945)-

Cell-free

preparations of the organism (Stadtman and Barker, 1949a) under anaerobic conditions quantitatively convert ethanol and acetate to butyrate.

Aerobically, with oxygen as an

electron acceptor, ethanol was oxidized to acetyl phosphate

10 and acetate.

A condensation of acetyl phosphate and

acetate was catalyzed and the product reduced to butyrate. Acetaldehyde proved to be an intermediate in the conver­ sion of ethanol to acetyl phosphate (Stadtman and Barker, 19^9b).

The conversion of acetyl phosphate plus acetate

to butyrate involves the uptake of two moles of hydrogen. Where the acetate concentration is low, caproic acid, as well as butyric, is formed.

In the presence of excess

acetate, the reaction goes almost quantitatively to buty­ rate (Stadtman and Barker, 19^9e).

Evidence obtained from

experiments with cell-free preparations indicated that acetoacetic acid is not the condensation product formed Ain the interaction between acetyl phosphate and acetate, *i since acetoacetate could be reduced by enzyme prepara­ tions only as far as beta hydroxy butyric acid (Stadtman and Barker, 19^9c).

The conversion of the condensation

product (whatever it might be) to butyrate should involve the formation of two intermediate products.

The first

should be in a state of oxidation corresponding to aceto­ acetate (but is not this compound).

The second should

correspond to crotonic acid, beta hydroxy butyric acid, or vinyl acetate.

Both crotonic acid and beta hydroxy

butyric acid were ruled out as possible intermediates. While vinyl acetate was shown not to be an obligatory intermediate in butyrate synthesis, this compound may

11 play a role in the conversion of acetyl phosphate and acetate to butyrate.

In the anaerobic oxidation of vinyl

acetate hydrogen is evolved.

This reaction leads to a net

production of phosphate bond energy in the form of acetyl phosphate.

It is probable that most of the energy for

synthesis of cell materials is derived from this or an analagous reaction.

The following represents the general­

ized mechanism for butyrate synthesis by Clostridium kluyverit ethanol + H^POjj.

>acetyl phosphate + 4h

acetyl phosphate + acetate C 4 compound + 4 h

»C^ compound + H^POjj.

» butyrate

Gur most detailed knowledge of aerobic dissimila­ tion of fatty acids by microorganisms comes from studies on the nature of rancidity in natural fats and oils.

The

rancidity is due to oxidative breakdown of fatty acids liberated after hydrolysis of lipids by various fungi.

In

many cases the oxidation is incomplete, and the products which accumulate, notably ketones, give characteristic odors and flavors associated with rancidity.

Starkle (1924)

was the first to discover the role of fungi in rancidity. He was able to isolate Peniclllium glaucum from rancid oil and showed that this mold, as well as Aspergillus niger and Aspergillus fumagatus, could form methyl ketones with one less carbon atom than was present in the fatty acids

12 on which the organisms were grown.

These early observa­

tions have since been confirmed by other workers (Stokoe, 1928; Coppock et al., 1928; Acklin,*1939; Thaler and Geist, 1939b; faufel et al., 1939)*

The ability of fungi to pro­

duce methyl ketones from fatty acids appears to be limited to the members of the series from (myristic).

(butyric) to C^ij.

Palmitic, stearic, and oleic acids are not

converted to ketones (Taufel et al., 1 9 3 6 ). Considerable speculation has surrounded the possible mechanisms involved.

Beta hydroxy acids give rise to

methyl ketones when attacked by molds which form ketones from fatty acids (Thaler and Geist, 1939b).

Thaler and

Eisenlohr (19^1) have shown that the reaction goes through alpha-beta unsaturated acids since these derivatives of butyric, caproic, capric, and myristic acids all yield methyl ketones.

More recently Thaler and Stahlin (1950)

have shown that Peniclllium glaucum cannot form methyl ketones from beta methyl— beta hydroxy acids, but with alpha methyl acids ketone formation occurs.

The following

sequence was suggested: saturated fatty acid— acid— acid

2 h-> alpha beta unsaturated

-tHgO— »beta hydroxy acid— --2H-»beta keto ►methyl ketone + carbon dioxide

Methyl ketones are not invariably mold products, since acids may be oxidized without a trace of ketone formation, and

13 yield of ketone is never quantitative.

Apparently two

mechanisms compete for beta keto acids— ketone formation and complete oxidation but that the long chain acids may be poisonous to the extent that they inhibit oxidative processes and ketone formation results (Stokoe, 1 9 2 8 ). Rahn (1 9 0 5 a, 1 9 0 5 b) was one of the first to study fat oxidation by bacteria.

In the course of other work

he showed that bacteria could grow on a medium with inor­ ganic nitrogen and stearic or palmitic acid as a carbon source.

Pozerski (1937a) showed that Escherichia coli could

use sodium stearate as a sole source of carbon for growth but that oleic acid could not serve a similar function (Pozerski 1937b).

Loebel, Shorr, and Richardson (1933)

studied metabolism of the tubercle bacillus on oleate, palmitate, and stearate and concluded that the acids could furnish energy but questioned whether they could be used to form protoplasm.

Bernheim (19^1) found that sodium salts

of fatty acids increased oxygen uptake of tubercle bacilli but that the effect was slight with the acids above valeric. At the same time Cutinelli (19^0) reported that monocarboxylic acids from ly* Gy to

to C5 were oxidized fairly rapid­

most rapidly and

to C2 0

speeds by the tuberculosis organism.

intermediate

Studying Pseudomonas

aeruginosa, Peppier (19^1) reported that the rate of oxygen uptake was greater on fatty acid substrates than on glycerol.

14 Attemonelli (1942) showed that Brucella melitensis, and several other species of the same genus, oxidized

to

C ^8 fatty acids*

Acetic and Cg to C-^g acids were most

readily attacked.

Yamaguchi (1946) found that sodium

salts of C-^ to Gj0 acids (except untested C1 3 ,

and

G^y) were readily oxidized by Pseudomonas aeruginosa and Micrococcus ochraceus.

Since the rate of oxidation was

generally greater with the fatty acids than with glucose, lactic and succinic acids, it was concluded that there is a close relationship between fatty acids and the normal metabolism of the cell.

Gray (1949), using Mycobacterium

tuberculosis, found that endogenous metabolism took place at the expense of stored lipid material.

Oglnsky, Smith,

and Solotorovsky (1950) reported that the oxidation of Cg to C1 0 acids by Mycobacterium tuberculosis var. avis was not affected by streptomycin but that of lauric, palmitic and stearic was partially inhibited.

Myristic

acid had an inhibitory effect on respiration.

It was sug­

gested that there may be two alternate pathways for fatty acid oxidation:

One for C2 to C1 0 and possibly to C^g, un­

affected by streptomycin, and one for C 1 2

cig sensitive

to streptomycin. Mechanisms of fatty acid oxidation in animals.

It

is apparent that our knowledge of fatty acid oxidation in

15 microorganisms is meager and that far more is known of anaerobic dissimilation processes than of aerobic metabolism, little or nothing is known of the actual mechanisms involved.

The problem of lipid metabolism

in animals has long concerned the biochemist.

While many

fundamental questions await answer, it would seem of value to review the various theories which have been pro­ posed for the mechanism of fatty acid oxidation in animal tissue.

A detailed review of the literature will not be

attempted, since excellent review articles on the subject have recently appeared (Breusch, 1948; Stadie, 1946). Beta oxidation, the earliest theory of fatty acid catabolism, was proposed by Knoop (1904) who fed phenyl derivatives of fatty acids to dogs and analyzed the ex­ cretion products in the urine.

He noted that when even

chain acids were fed hippuric acid was invariably excreted in the urine; odd chain acids yielded phenaceturic acid. Knoop made no assumptions concerning the possible splitting off of two-carbon fragments or whether the oxidations were successive or simultaneous.

The hypothesis came to mean

that each fatty acid molecule is oxidized through a series of fatty acids, each shorter by two carbon atoms than its immediate precursor.

Finally one molecule of

butyric acid remains, and this in turn is oxidized to acetoacetic acid.

The balance of the molecule is converted

16 presumably to aeetic acid molecules. The original hypothesis of Knoop did not explain, however, the great capacity of the liver to form acetone bodies, since it allowed for the formation of only a single acetone body per fatty acid molecule.

Further, the

hypothetical acetic acid molecules were never isolated nor were fatty acids of intermediate chain length found in the tissues and fat deposits of mammals.

On this basis, a

second theory of oxidation was formulated (Jowett and Quastel, 1935; Butts, et al., 1935)*

According to their

theory of multiple alternate oxidation, oxidation begins at the beta carbon atom and simultaneously at each alternate carbon atom along the whole length of the carbon chain.

Ac­

cordingly, fatty acids are oxidized first to polyketo com­ pounds, and following this preliminary oxidative step, the molecule splits into a number of acetoacetic acid molecules, the number depending upon the length of the chain.

This

then accounted for the observed increase in acetone body production with increased chain length.

Breusch (19^8)

believes that this mechanism is the major pathway of fatty acid oxidation in the liver but not in the muscle or kidney tissue. In the mechanism of multiple alternate oxidation it was implied that long chain polyketo acids were formed and that these then split into four-carbon units forming ketone

17 bodies.

The question arose as to the origin of ketone

bodies from fatty acids with carbon atoms not an even multiple of four, since these also are converted into ketone bodies.

Likewise, odd chain acids are to some

extent ketogenie.

The sole postulate offered was that

ketone bodies might arise from C 2 condensations.

The

theory of successive beta oxidation— condensation was formulated by McKay (19^3)*

the fatty acid molecule, re­

gardless of its nature, is split into C2 units which are then re-assembled into ketone bodies.

The sole difference

between this theory and that of multiple alternate oxida­ tion is that the fatty acids are split on the one hand in­ to four-carbon units and on the other into two-carbon fragments which are re-assembled into ketones (four-carbon units).

There is experimental evidence to support both

of these theories, and the possibility exists that both mechanisms occur in the body (Stadie, 1946). Verkade and van der Lee (1932) noted that trigly­ cerides of saturated fatty acids of medium chain length (C8 to

) are partially excreted in the urine as di-

carboxylic acids.

The evidence indicated that the fatty

acid molecule was oxidized first at the omega carbon atom and then simultaneously beta oxidation occurred at both ends of the chain, since capric acid (C10) yielded di­ carboxyl ic acids with ten, eight, and six carbon atoms.

18 Experimental evidence Indicated that with fatty acids of intermediate chain length omega oxidation competes with beta oxidation, with about 9 0 per cent of the acid follow­ ing the latter pathway*

The mechanism of omega oxidation

is of little importance in animal nutrition, since most of the catabolism of fatty acids apparently occurs in the liver by the multiple alternate route, and the inter­ mediate acids do not accumulate (Breusch, 1948). Whatever the mechanism of primary attack, fragments are formed which must undergo further oxidation.

The pres­

ent evidence indicates that products of the primary oxida­ tion undergo terminal degradation in a manner similar to carbohydrates.

Breusch (1948) has summarized as follows:

"Acetoacetic acid, the primary metabolite of fatty acids in the liver, and to a smaller extent, higher beta keto fatty acids eventually formed in muscle tissue, undergo condensation with oxalacetic acid in muscle and kidney tissue, resulting in the formation of Cg-tricarbosylic acids due to the action of an enzyme system (citrogenase). The acids, citric acid=^==i: cis-aconitic acid = = ^ i s o citric acid, are in enzymatic equilibrium with one another, of which citric acid makes up 80 per cent.

These acids

lose two carbon atoms through oxidation and then are readi­ ly oxidized to oxalacetic acid (C^), which in turn under­ goes new condensations and thus burns fatty acids at the

19 rate of two-carbon fragments; this is called the C$tricarboxylic acid cycle."

The reactions involved may be

represented as follows:

R.

I 0 =0

O^C I

CHa 1

COOH

CHt

I COOH

C

COOH

C O O H CO O H

JiJ-K.e’fco F a .t'ty A c i d

C OOH

Hypo'the.fcica.I I n t crH«dia.t«-

O xal a c e t ic A c id

OH

ft COOH FaCCy A c id I 0.001 M DNP

256

257 272 252

81.5 82.0 87.0 80.3

Substrate concentrations: acetic acid, 2.0 micromole per 2 . 0 ml fluid in flask; all other acids, 0.4 or 0 . 6 micro­ mole per 2 . 0 ml fluid in flask, depending on particular experiment. Experiments conducted in air atmosphere, with 10 per cent KOH to absorb COg; pH 7-0; 30°C.

76 to theoretical amounts for complete oxidation decreased with increasing chain length, the percentage of theoretical uptake being about the same for eaprylic, pelargonic, and capric acids.

Cells adapted to acetic and pelargonic acids

showed greater uptake on the homologous acids than did the unadapted cells; with cells adapted to heptylic, caprylie, and capric acids the reverse effect was noted.

It is ap­

parent that oxidative assimilation occurs with all the acids tested and that the amount of assimilation increases with molecular weight of the fatty acids. In preliminary experiments with capric acid oxida­ tion, it was found that DNP increased oxygen uptake by both adapted and unadapted cells.

Caprate oxidation was

most nearly complete in the presence of 0.00075 M DNP; 0 .0 0 0 5 M and 0 . 0 0 1 M concentrations gave slightly lower

values.

In most cases tests were conducted with 0.0005

and 0.00075 M DNP. DNP completely inhibited the oxidation of caproic acid by both adapted and unadapted cells.

Metabolism of

acetic acid by C^Q-grown cells (and thus cells simultan­ eously adapted to acetate) was completely inhibited; unadapted cells and cells exposed to acetic acid showed a very low level of oxygen uptake in the presence of DNP. It seems probable that no actual utilization of acetate occurred under these conditions, the values in excess of

77 autorespiratory uptake perhaps representing a slight stim­ ulation of endogenous metabolism.

As a working hypothesis

it may be assumed that DNP at pH 7.0 blocks acetate and caproate metabolism in Serratia marcescens (Alphin). Reference to Figure 8 and Table III shows that DNP brings about both quantitative and qualitative changes in the oxidation of heptylate.

Unadapted cells show no

oxygen uptake in excess of the autorespiratory level, while cells grown on capric acid-mineral salts medium (and thus adapted to heptylic acid) show an oxygen uptake significantly in excess of endogenous but at a slow rate as compared with adapted cells in the absence of cell poison.

It will be noted that CjiQ-grown cells showed

a greater uptake (75 }il perjim) than did the Cj-exposed cells (23 jal per jam).

In the experiment with C^Q-grown

cells 0.00075 M DNP was used; while 0.0005 M DNP was used with the exposed organisms.

Whether this difference in

concentration of poison accounts for the observed differ­ ence in uptake is not apparent.

In both cases, the rate

was so slow it was necessary to subtract autorespiration directly from total uptake; this may be an important factor. DNP brought about a marked inhibition of caprylate and pelargonate oxidation by unadapted cells.

Uptake in

both cases was low as compared with that for unadapted

78 cells in the absence of DNP (see Figure 8 ).

With pelargon­

ic acid it is questionable whether the uptake above the endogenous level represents utilization of substrate.

It

must be emphasized that in repeated experiments with oxida­ tion of pelargonate by unadapted cells the rate was always slightly in excess of autorespiratory uptake, even with low concentrations of DNP. Unadapted cells show a higher level of oxygen uptake on caprylate than pelargonate in the presence of DNP.

It is, nevertheless, at a slower rate and

the net uptake is less than that with the same cells in the absence of poison.

DNP had no inhibitory effect on caprylic

acid oxidation when Serratia (Alphin) cells were grown on capric acid medium and then tested for caprylate oxidation in the presence of DNP.

The net oxygen uptake was in excess

of that in the absence of poison, being approximately 8 6 per cent of the theoretical value for complete oxidation.

No

data are presented for caprylate-grown or exposed cells; in preliminary studies it was found that DNP did not inhibit caprylic acid oxidation in these cells, the rate and quan­ titative uptake being comparable to that for caprie-grown cells.

Similarly, DNP had no inhibitory effect on pelar­

gonate oxidation by cells exposed to pelargonic acid or grown on capric acid-mineral salts medium.

With C^-exposed

cells the uptake was essentially the same as that for the exposed cells in the absence of poison, but with the ° 1 0 "

79 grown cells the total uptake was Increased, approximately 8 5 per cent of the theoretical amount of oxygen being

consumed.

It will be noted that 0.0005 M DNP was used

with the exposed cells, 0.00075 M DNP with the caprategrown cells.

In many experiments with pelargonic acid

oxidation it was noted that cells adapted to capric acid showed greater uptake on pelargonic acid than did cells specifically adapted to pelargonic acid.

One might argue

that in the absence of cell poisons the cells specifically adapted to the odd-chain acid can more efficiently as­ similate that acid than can the caprate-adapted cells. In the presence of cell poisons this assimilation is presumably inhibited, and the divergence between results obtained with the two types of adapted cells seems to have no logical explanation. The reactions of Serratia marcescens cells -so capric acid were unlike the reactions for the lower acids.

DNP

did not inhibit oxidation by either adapted or unadapted cells.

Total oxygen uptake with unadapted cells, with

C10-grown cells, and with C^Q-exposed cells was always in excess of the value obtained in the absence of DNP.

C10-

grown cells showed 8 7 per cent of the theoretical uptake for complete oxidation, unadapted cells 9 9 per cent, and C10-exposed cells 8 1 . 5 per cent.

The unadapted cells

utilized the acid only after an initial lag period, just

80 as did the unadapted cells in the absence of DNP.

Further,

if unadapted cells were allowed to oxidize capric acid to completion and then were exposed to fresh substrate (poured from the side-arm) the oxidation once again proceeded with an initial lag period.

Such cells also showed a lag period

in the oxidation of other fatty acids.

It will be recalled

that cells allowed to oxidize capric acid in the absence of DNP subsequently oxidized caprate and all the other acids tested with no lag period. Neither undecylic nor tridecylic acids were oxidized by unadapted cells in the presence of DNP (Table IV); under these conditions there was a slow rate of oxygen uptake slightly above the autorespiratory uptake, similar to that observed in the oxidation of pelargonic acid under the same conditions.

Laurie and myristic acids were oxidized

by the unadapted cells In the presence of DNP, but the rate of oxygen uptake was only about a fourth of that observed In the absence of the cell poison; the amount of oxygen consumed was much less than that taken up by unadapted cells without DNP.

When C^Q-grown cells were used, the

C n to Ci 4 acids were all oxidized in the presence of DNP. No quantitative data on the extent of oxidation of these acids were obtained. Sodium azide in 0.06 M concentration had effects similar to 0.00075 M DNP, but the over-all oxygen uptake

81 TABLE IV THE EFFECT OF pH ON THE OXIDATION OF FATTY ACIDS BY SERRATIA (ALPHIN) CELLS IN THE PRESENCE AND ABSENCE OF 2:4-DINITROPHENOL SubpH T.O1 pH 8.0 strate^ Unadapted Adapted Unadapted Adapted Unadapted Adapted DNP DNP DNP DNP _ *

_ *

/

/

/ (93:6)

/

c6

-

-

-

-

-

-

°7

-

-

/

-

/ (6 0 #)

c8

_ *

/

/

/

/ (74#)

_■*

/

/

/

/ (64#)

/

/

/

/

/ (9 8 #)

/ (74#)

_•#

/

/

/

_ *

/

/

/

/

/

/

/

/

/

/

°2

c9 ClO C11 C12

c13 c14

^** _ *

**

(

62$

/ _ *

/ /

1 All substrates oxidized by Serratia (Alphin) at pH 7*0 in

absence of DNP. 2 Acetic acid flask concentration— 0.001 M; all other acids—

0.0002 M. * Oxygen uptake slightly above endogenous. ** Rate of oxygen uptake lower than that in the absence of DNP; total oxygen uptake less than when DNP absent. Figures in parenthesis represent percentage of theoretical oxygen uptake for complete oxidation to carbon dioxide and water. All experiments at 30°C., air atmosphere with 10 per cent KOH to absorb carbon dioxide. Unadapted— glucose-grown Serratia (Alphin) cells. Adapted— caprate-grown Serratia (Alphin) cells.

)

82

was not as great as in the presence of the phenolic com­ pound.

Azide inhibited acetate oxidation to the same

extent as did DNP.

Caproate and heptylate oxidation

were not studied in the presence of azide.

The azide

increased oxygen uptake on caprylic, pelargonic, and capric acids when adapted cells were used but not to the same extent as did DNP.

With unadapted cells, azide had

no inhibitory effect on caprate oxidation; the oxidation of pelargonate was inhibited to the same extent as was noted with DNP.

No data were obtained for unadapted cells

in the presence of azide and caprylate. The data indicate that the oxidation of certain acids is chemically blocked by DNP.

With acetic and

caproic acids, for instance, cells completely adapted to these compounds fail to metabolize them in the presence of DNP.

It appears likely that adaptation is also in­

hibited, but the data do not conclusively prove this, i.e. adaptation (whatever it may involve) might occur in the presence of DNP, but the presence of DNP also might pre­ vent its observation.

No attempts have yet been made to

determine whether adaptation can occur without fatty acid oxidation. The oxidation of certain other acids, caprylic and pelargonic for instance, by unadapted cells is prevented by DNP, but such oxidation by adapted cells is unaffected

83 by DNP.

This indicates that DNP prevents an adaptation pro­

cess which normally occurs when glucose-grown cells are exposed to fatty acids.

Whether this adaptation process

involves the formation of adaptive enzymes has not been determined. Only in the presence of capric acid was the amount of oxygen consumption indicative of complete oxidation. This occurred only when glucose-grown cells were used; caprate-grown cells consumed only

8

j

per cent of the amount

of oxygen required for dissimilation of cparic acid to carbon dioxide and water.

Since it is improbable that the

oxidative pathways are different in the two types of cells, it appears likely that the lower uptake with adapted cells is due to oxidative assimilation.

This suggests that a

higher concentration of DNP is necessary to inhibit com­ pletely oxidative assimilation in adapted cells.

A similar

effect was noted in the study of acetic and capric acid oxidation at pH 8.0 (see Table IV). Pelargonate oxidation by caprate-grown cells result­ ed in the uptake of

237

jul of oxygen per jam of substrate—

85 per cent of theoretical.

This value is quite close to

that observed in the oxidation of caprate by the same cells (8 7

per cent).

Since pelargonate oxidation was inhibited

by DNP in unadapted cells, these data do not permit the conclusion that the failure to show theoretical oxygen

84 uptake is due to assimilation rather than incomplete oxida­ tion.

If an unoxidized fragment were to accumulate during

the oxidation of pelargonate in the presence of DNP, this substance should be in a state of oxidation corresponding to acetic acid.

The oxidation of pelargonic acid with the

accumulation of one mole of acetic acid per mole of pelargon ic acid would require the consumption of 2 3 5 pi of oxygen per pm of pelargonate.

Since acetate is not oxidized at

pH 7-0 in the presence of DNP, the results suggest that acetate may accumulate during the oxidation of the ninecarbon acid.

Data to be presented later in this chapter

indicate that this is not the case and that it is probable that the apparent incomplete oxidation is due to oxidative assimilation. Oxidation of caprylic acid resulted in the uptake of 2 1 0 pi of oxygen per p m of acid when caprate-grown cells were used— 8 5 - 6 per cent of the theoretical amount for complete oxidation.

As with pelargonate, oxidation by un­

adapted cells was inhibited by DNP.

The percentage of

theoretical uptake with caprate-grown cells is sufficiently close to that observed in pelargonate and caprate oxidation by the same organisms to allow the assumption that DNP is only about 8 5 per cent effective as an inhibitor of oxida­ tive assimilation in caprate-grown cells. Since neither heptylate nor caproate were appreciably

85 oxidized in the presence of DNP at pH 7.0, indications as to the extent of oxidation of these acids can be obtained only from the data obtained in the absence of DNP.

Under

these conditions, caprate-grown cells consumed 1 5 0 yil of oxygen per jam of heptylic acid.

Without considering the

effect of assimilation on the observed uptake, this in­ dicates oxidation beyond the propionic acid stage.

Similar­

ly, unadapted cells showed an uptake of 1 3 0 jil of oxygen per jam of caproate, indicating disinflation at least to a stage of oxidation corresponding to acetate.

Since work

with the higher acids indicated that in the absence of DNP 30-40 per cent of the substrate is assimilated, it is prob­ able that caproate is oxidized beyond the acetate stage, possibly to carbon dioxide and water. The oxidation of fatty acids at pH 8 ._0 i n the pres­ ence and absence of DNP. Unlike sodium azide, the effective­ ness of DNP as an inhibitor of oxidative assimilation is in direct relationship to the hydrogen ion concentration (Doudoroff, 1940).

The preceding work indicated that

0.00075 M DNP at pH 7-0 might exert at least two distinct inhibitory effects on fatty acid oxidation by Serratia (Alphin), i.e. direct blockage of the enzymes concerned with oxidation and inhibition of adaptation to fatty acid oxidation.

It seemed possible that two different concentra­

tion of DNP might be responsible for the inhibitory actions,

86

one for inhibition of oxidation and one for inhibition of adaptation.

Experiments were conducted in which the oxida­

tion of fatty acids was studied at pH 8.0, both in the presence and in the absence of DNP. The results summarized in Table IV show that with the exception of caproic acid, all acids oxidized at pH 7*0 were also oxidized at pH 8.0.

Heptylic acid is oxidized by cells

previously adapted to heptylate oxidation by growth on capric acid medium but is not oxidized by unadapted cells.

Failure

to oxidize caproic acid under any of the conditions imposed at pH 8.0 indicates that at this pH the acid may become toxic to the cell; at the higher pH the ionic equilibrium shifts to a higher concentration of the salt of caproic acid and away from the free acid.

Alternatively, there may

be something unique in the metabolism of caproic acid (as compared to the other acids tested) in that its oxidation is blocked at a pH at which the other acids are metabolized. At pH 8.0 DNP prevents oxidation of heptylate, caprylate, pelargonate, undecylate, and tridecylate by un­ adapted cells.

C 2 9 Gio* C 1 2 9 anci cl4 acids are oxidized

by unadapted cells in the presence of DNP.

Most notable

is the fact that acetic acid is nearly completely oxidized in the presence of DNP at pH 8.0.

It will be recalled that

acetate oxidation was blocked by DNP in both adapted and unadapted cells at pH 7-0.

87 With the exception of caproic acid, all the acids are oxidized by adapted cells (Cio-grown) in the presence of DNP at pH 8.0.

Quantitatively, the amount of oxygen

consumed under these conditions is less than at pH 7.0. Oxygen uptake on acetic and heptylic acids is actually less than that observed when adapted cells oxidize these acids at pH 7*0 in the absence of DNP.

With the other

acids, uptake is greater than that for adapted cells at pH 7-0 in the absence of DNP but is less than that by or­ ganisms at the lower pH in the presence of the phenolic compound.

Further, heptylic acid oxidation is completely

inhibited by DNP at the lower pH, but the acid is oxidized by adapted cells at pH 8.0.

Thus DNP is not as effective

as an inhibitor of oxidative assimilation at pH 8.0.

Thus

DNP is not as effective as an inhibitor of oxidative as­ similation at pH 8.0 as at pH 7*0. Difficult to explain is the fact that neither capric nor acetic acids are oxidized to completion by adapted cells at pH 8.0 in the presence of DNP; unadapted cells show oxygen uptake indicating complete oxidation to carbon dioxide and water under the same conditions.

This suggests

that a higher concentration of DNP may be required to in­ hibit oxidative assimilation in adapted cells.

A similar

finding was reported in the previous section— at pH 7*0 in the presence of DNP capric acid was completely oxidized

88

by unadapted cells ( 9 9 per cent) but showed only 8 7 per cent of theoretical oxygen consumption with C^Q-grown organisms.

The age of the cells may account for these

differences, since the Cxo-grown cells were harvested from 40 hour cultures and the glucose-grown cells from 18 hour cultures.

It is often stated that poisonous sub­

stances are most effective against young cells.

The fact

that Cio-exposed cells (18 hour glucose-grown cells) show­ ed less uptake than C^Q-grown cells at pH 7*0 would In­ dicate that perhaps the differences in uptake between adap­ ted cells and unadapted cells is due to something other than the age of the organisms. If one studies the patterns of oxidation at the two pH levels, it will be noted that the odd chain acids--C1 3 , C-q, and C^--gave identical patterns; the even chain acid, caprylic, shows the same pattern.

and C^q acids

have similar patterns; here, however, DNP had some Inhibit­ ory effect on the oxidation of lauric and myristic acids by unadapted cells.

The rate was significantly decreased

and also the total oxygen uptake. is unaffected by DNP.

Capric acid oxidation

Acetic, caproic, and heptylic acids

gave patterns unlike the higher acids and also differed from one another. be discussed later.

The significance of these findings will

89 Evidence that pelargonic and capric acids are completely oxidized at pH 7-0 _in the presence of DNP.

It will

be recalled that in the presence of DNP (0.00075 M) un­ adapted cells showed 9 9 per cent of the theoretical oxygen uptake for complete oxidation of capric acid.

C^Q-exposed

cells showed 87 per cent of the calculated amount.

Similar­

ly, C10-grown cells consumed only 8 5 per cent of the amount of oxygen required for complete oxidation of pelargonic aeid.

Analysis of the data indicated that if the uptakes

below theoretical were due to incomplete oxidation, the un­ oxidized fragments should correspond closely to acetic acid.

Since acetic acid is not oxidized under these condi­

tions, it seemed possible that it might accumulate.

It was

found, however, that acetate was metabolized at pH 8.0 in the presence of DNP.

Experiments were conducted in which

capric acid was oxidized by adapted cells at pH 7-0 in the presence of DNP.

When the autorespiratory level was reach­

ed, alkaline buffer was poured from the side-arm and the pH In the flask was thus raised to 8.0.

Similarly, pelargon­

ic acid was oxidized by C^Q-grown cells at pH 7-0 and then the pH raised to 8.0 in the same manner.

In both cases,

oxygen uptake was followed to the point at which endogenous respiration started at the lower pH, and then the uptake was measured after addition of the buffer solution.

In

neither case was there any further oxygen uptake above the

90 autorespiratory level after the pH was raised.

If acetic

acid had accumulated during the oxidation at pH 7.0, one would have expected further uptake of oxygen after the pH had been raised, since acetate is metabolized at the high­ er pH.

Since there was no further uptake above the endo­

genous level, the results indicate that acetate does not accumulate in either pelargonate pH 7-0 in the presence of DNP.

:or caprate oxidation at Since unadapted cells had

previously been shown to oxidize caprate to completion at pH 7-0, it appears reasonable to assume that the apparent incomplete oxidation of pelargonate, caprate, and caprylate is due to incomplete inhibition of oxidative assimilation.

DISCUSSION Work with cell poisons gives evidence, in some cases indirect, that the C2 > Cg, Cj ,

Cg, Gg, and C ^ 0 fatty acids

are completely oxidized to carbon dioxide and water by cell suspensions of Serratia (Alphin). With capric acid 9 9 per cent of theoretical oxygen uptake was obtained with

unadapted cells in the presence of 0.00075 M DNP at pH 7«0. This was the only case in which unequivocal proof for complete oxidation was obtained.

With acetic acid 93 per

cent of the theoretical uptake was observed in the presence of DNP with unadapted cells at pH 8.0; it is assumed that

91 failure to observe the consumption of theoretical amount oxygen under these conditions is due in part to experi­ mental error and in part to the failure of DNP to inhibit completely oxidative assimilation at this pH.

Caprate-

grown cells showed approximately 8 5 per cent of the oxygen uptake necessary for complete oxidation of caprylic and pelargonic acids in the presence of DNP; indirect evidence indicated that this value represents the effectiveness of DNP as an inhibitor of oxidative assimilation rather than incomplete oxidation of the eight and nine-carbon acids. Since neither heptylic nor caproic acid was oxidized in the presence of DNP at pH 7*0, it was possible to obtain evidence as to the extent of oxidation of these acids only from quantitative results obtained in the absence of DNP. Such data indicated that in the absence of DNP oxidation of heptylate proceeds beyond the propionic acid stage and the oxidation of caproate beyond acetic acid.

When one

takes into account the fact that 3 0 - ^ 0 P©** cent of the available substrate is assimilated, it appears likely that both these acids are completely oxidized.

No quantitative

data were obtained on the extent to which the higher acids are oxidized. In previous studies it was found that Serratia (Alphin) cells do not oxidize butyric, valeric, or pro­ pionic acids.

The quantitative data indicate that the

oxidation of the C 6 > Oq , and G^q acids proceeds beyond the butyric acid stage and that the C7 and Cg acids are oxidiz­ ed beyond the propionic acid stage.

This indicates that

butyric acid is probably not an intermediate in the oxida­ tion of higher even chain acids and that neither propionic nor valeric acids are intermediates in the oxidation of higher odd chain acids.

Since the data show that oxida­

tion goes beyond the propionic and butyric acid stage, it is not likely that the C3 , 0 4 , or the oxidation of higher acids.

acids accumulate in

According to the classical

theory of beta oxidation, butyrate should be an intermediate in the oxidation of higher even chain acids and propionate in the oxidation of higher odd chain acids (Breusch, 1948). If one assumes that the repeated failures to demonstrate oxidation of butyric, propionic, and valeric acids is due to lack of appropriate enzymes in the cell for the metabol­ ism of these compounds, then beta oxidation is not the mechanism for attack on C6 to C 1 0 fatty acids.

If, on the

other hand, this failure to observe oxidation is due to impermeability of the cells to these compounds, and if the appropriate enzymes for the oxidation of these acids are present in the cells, then it is possible that the three, four, and five-carbon acids are intermediates and that beta oxidation occurs.

The only feasible manner in which

this problem can be solved is through studies with cell-free

93 enzyme preparations.

Under these conditions, permeability

effects should be eliminated, and if the enzymes for the oxidation of

fatty acids are present in the cells,

they should be as readily demonstrated as those for the higher acids.

Should cell-free preparations capable of

catalyzing the oxidation of the higher acids fail to activate oxidation of

acids, then it would have to

be concluded that some mechanism other than beta oxida­ tion occurs.

This problem will be more completely dis­

cussed in the following chapter. The quantitative data on oxygen uptake by unadapted cells in the presence of DNP at pH 7*0 indicated that capric acid is completely oxidized to carbon dioxide and water.

Under the same conditions, unadapted cells fail

to oxidize pelargonic, caprylic, heptylic, caproic, and acetic acids.

Thus, the nine, eight, seven, six and two-

carbon fatty acids cannot be direct intermediates in the oxidation of caprate by Serratia cells.

Indirect evidence

suggested that adapted cells completely oxidize capric, pelargonic, and caprylic acids at pH 7-0 in the presence of DNP; but the same cells fail to oxidize heptylic, caproic, and acetic acids in the presence of DNP.

Hence,

neither heptylate,' caproate, nor acetate can be direct intermediates in the oxidation of caprylic and pelargonic acids.

Since the technique of simultaneous adaptation

9^

indicated a close relationship between the enzymes catalyzing the primary steps in the oxidation of all these acids, the results suggest that DNP interferes with a reaction necessary for activation of heptylate, caproate, and acetate oxidation.

The nature of this reaction is not

apparent.. Cross et a l . (19^9) have shown that DNP inhibits a reaction involving the transformation of phosphate; this phosphate transformation is necessary for the activation of the oxidation of certain lower fatty acids by the cyclophorase system.

It may be that a similar reaction is neces­

sary for the activation of oxidation of acetate, caproate, and heptylate.

If such is the case, then "activated” acet­

ate and caproate may be intermediates in the oxidation of caprate and caprylate, and "activated" heptylate may be an intermediate In the oxidation of pelargonate. DNP inhibited oxidation of acetate, caproate, heptylate, caprylate, pelargonate, undecylate, and tridecylate in unadapted cells at pH 7«0.

The same concen­

tration of DNP did not interfere with the oxidation of caprylate, pelargonate, undecylate, and tridecylate in adapted cells.

At pH 8.0 DNP failed to inhibit acetate

oxidation in either adapted or unadapted cells; heptylate was oxidized by adapted cells.

This inhibition Is ap­

parently a blocking of the adaptation process (whatever it involves), since cells adapted to the oxidation of

95 these acids (by growth on caprate) were able to oxidize the fatty acids in the presence of DNP.

Caproate appears

to be the only exception, since it was not oxidized under any of the conditions imposed when DNP was present. Monod (1944), Reiner (1946), and Spiegleman (1 9 4 7 ) have indicated that DNP inhibits the formation of adaptive enzymes in microorganisms.

This fact would lead one to the

conclusion that the Cy, Cq, C9 , C-q, and C f a t t y aeids are attacked through adaptive enzymes, while the

G±o>

C12, and C a c i d s are catalyzed by reactions involving only constitutive enzymes.

The type of attack on caproic

acid might be either adaptive or constitutive, since DNP apparently interferes directly with some enzymatic step in its oxidation.

On the basis of experiments with simul­

taneous adaptation (Chapter III) it appears unlikely that some of the acids are attacked adaptively and others constitutively.

Exposure of cells to any of the fatty acids

known to be oxidized by the Serratia produces organisms which have no significant lag period in the oxidation of the other acids.

If certain acids were oxidized by adap­

tive enzymes and others by constitutive enzymes, one could not expect this reciprocal adaptation to occur.

It is

probable that the enzymes involved in the oxidation of these acids are either all adaptive or all constitutive. It will be recalled that despite the fact that caprate was

96 oxidized by unadapted cells in the presence of DNP, these cells did not become adapted to any of the acids, including capric acid.

When such organisms were subsequently exposed

to capric acid, oxidation proceeded after an initial lag period characteristic of unadapted cells*

While the un­

adapted cells are capable of caprate oxidation in the presence of DNP, the process of adaptation by such cells to other acids is blocked; in fact even adaptation to capric acid is prevented (if elimination of the lag period is to be considered the criterion for adaptation).

Since

DNP inhibits oxidative assimilation, it is probable that the adaptation process involves utilization of exogenously supplied substrate.

In the presence of DNP, this process

of assimilation is blocked, and although certain acids may be oxidized by unadapted cells in the presence of DNP, und­ er these conditions there is no adaptation.

It is not pos­

sible to say on the basis of the present information whether adaptation is a process involving the formation of adaptive enzymes.

The only logical approach to this problem appears

to be through preparation of cell-free enzyme systems from both adapted and unadapted cells. The failure of both adapted and unadapted cells to oxidize caproic acid in the presence of DNP suggests that some reaction essential for the oxidation of the six-carbon acid is blocked.

While it appears unlikely that this acid

97 is oxidized in a different manner than all the other acids, the results with DNP, together with the failure of cells to oxidize caproate at pH 8.0, suggest this possibility. The unique results from experiments with caproic acid bear further investigation.

SUMMARY The effects of 2:4-dinitrophenol and sodium azide on fatty acid oxidation by Serratia (Alphin) were studied. Both substances inhibited oxidative assimilation at pH 7.0, the former compound proving most effective in this respect. On the basis of direct and indirect evidence from the experi­ ments with DNP it was concluded that capric, pelargonic, caprylic, and acetic acids are completely oxidized by the Serratia cells.

There was good evidence to indicate that

heptylic and caproic acids are also completely metabolized without the accumulation of intermediate substances.

At pH

7.0 appropriate concentrations of DNP prevented the oxida­ tion of acetic, caproic, heptylic, caprylic, pelargonic, undecylic, and tridecylic acids by glucose-grown cells. Under the same conditions, capric, lauric, and myristic acids were oxidized after lag periods.

Caprate-grown cells

oxidized the eight, nine, and thirteen-carbon acids in the presence of DNP at the same pH.

At a pH of 8.0 acetic acid

98 was oxidized by both types of cells and heptylic acid by caprate-grown cells only.

The reactions of the other

acids were the same as that at the lower pH.

Under no

circumstances was caproate oxidized in the presence of DNP in the concentrations used.

CHAPTER V STUDIES ON THE RELATIONSHIP OF VARIOUS FATTY ACID DERIVATIVES TO THE PRIMARY OXIDATIVE REACTIONS IN FATTY ACID OXIDATION All proposed mechanisms for fatty acid catabolism involve the formation of beta keto derivatives of fatty acids.

If it is assumed that beta keto acids are key com­

pounds in fat metabolism, then the question arises as to the steps involved in the conversion of a saturated acid to its beta keto derivative.

The oxidation of a saturated

acid to the corresponding keto acid should result in the net removal of four hydrogen atoms.

According to current

concepts of biological oxidations, such a chain of reac­ tions should involve at least two intermediate steps, two hydrogen atoms being removed in each step.

Various theories

have been proposed for the mechanism of ketone formation (Breusch, 1948; Foster, 1949).

While absolute proof is

lacking, evidence indicates that the following sequence occurs both in animal tissues and in molds: R -C H ., CHaCOOH

-PH

----------- *

H H R -C =C -C O O H

+ H.»f"S -— >

OH H R -C -C -C O O H

Fatty acid

H H

_pu ---------------- .

o ^

.. H R - C - C - CO O H H

From the standpoint of comparative biochemistry, the sequence is analagous to the Thunberg-Wieland

mechanism for the

100

oxidation of succinic to oxalacetic acid and to the primary steps in the oxidative deamination of amino acids. The present study concerned itself with the rela­ tionship of the alpha-beta unsaturated, beta hydroxy, and beta keto derivatives of capric acid to the metabolism of capric acid and other fatty acids.

EXPERIMENTAL Solutions of the potassium salts of beta hydroxy1 and beta keto2 capric acid were prepared in M/20 phosphate buffer.

Alpha-beta unsaturated capric acid^ was added to

M/20 phosphate buffer to give a 0.01 M solution; the pH was adjusted to 7 * 0 by the addition of potassium hydroxide. For manometric studies, 0.0006 M solutions of the three caprate derivatives were prepared.

Oxygen uptake in the

presence of these compounds was measured in the manner described in previous chapters,using 1 . 0

ml of diluted

stock solution in the side-arm and 1 . 0 ml of cells in the main well.

Cell suspensions were prepared by washing 16

to 20 hour glucose-grown or 40 hour caprate-grown Serratia marcescens (Alphin) cells three times in M/20 phosphate

1 Prepared according to the method of Thaler and Geist (1939b). 2 Prepared

according to the method of Stenhagen (19^3)-

3 Prepared according to the method of Tulus (19^*0 •

101

buffer.

All suspensions were adjusted to a standard

turbidity as measured on the Klett-Summerson apparatus. In most cases, oxygen uptake was measured until sub­ strate had been completely utilized, but in certain in­ stances experiments were stopped as soon as it was pos­ sible to establish the shape of the oxidation curve. The oxidation of caprate derivatives by glucose and caprate-grown cells * Curves for glucose-grown cells in Figures 10, 11, and 12 show that the alpha-beta unsaturat­ ed, the beta hydroxy, and the beta keto derivatives of capric acid were oxidized after lag periods of 2 6 , 40, and 19 minutes, respectively.

The same cells showed a 30

minute lag in the oxidation of capric acid.

The lag periods

were estimated by extension of the steepest part of the curve to the time axis. Cells harvested from capric acid medium showed no lag in the oxidation of beta keto capric acid.

The lag

periods in the oxidation of the unsaturated and hydroxy derivatives were shortened but not eliminated, being ap­ proximately 8 minutes in each case.

The caprate-grown

cells showed a 4 minute lag in the oxidation of capric acid. If one assumes that the lag periods represent a time during which adaptive enzymes are being formed, then

102

\

I

©O

•10 &r ^Wri

80TO-

20

40

60

80

120

140

160

160

200

T I M E IN M I N U T E *

M/20 phosphate buffer, pH 7.0, 30°C. Experiments conducted in air atmosphere with 0.1 ml KOH in center well. Flaskconcentration of alpha-beta un­ saturated capric acid--0.0003 M. Glucose-grown cells used for "exposure." FIGURE 9 OXIDATION OF ALPHA-BETA UNSATURATED CAPRIC ACID BY SERRATIA (ALPHIN)

103

no- -

IOO--

90- -

80- -

un»atur«,tcd expo t e d

Z

ui cr

> * o

60-

30 -

20-

20

60

80

100

ISO

HO

160

180

200

TIME IN MINUTES

M/20 phosphate buffer, pH 7*0, 30°C. Experiments conducted in air atmosphere with 0.1 ml KOH in center well. Flask concentration of beta hydroxy capric acid— 0.0003 M. Glucose-grown cells used for exposure.

FIGURE 10 OXIDATION OF BETA HYDROXY CAPRIC ACID BY SERRATIA (ALPHIN)

104

IIOt

C|o 8rown

100-■ 9 0 --

7 0 --

-**

30-

1040

60

80 T IM E

100

IN

20

160

ISO

500

M IN U T E S

M/20 phosphate buffer, pH 7*0, 30°C. Experiments conducted in air atmosphere with 0.1 ml KOH in center well. Flask concentration beta keto capric acid— 0.0003 M. Glucose-grown cells used for "exposure. 11 FIGURE 11 OXIDATION OF BETA KETO CAPRIC ACID BY SERRATIA (ALPHIN)

105 according to the theory developed by Stanier (1947) it must be concluded that beta keto capric acid is an inter­ mediate in the oxidation of capric acid.

While the lag

periods in the oxidation of the unsaturated and hydroxy derivatives of capric acid were not completely eliminated as a result of growth on capric acid medium, the same cells showed a 4 minute lag period in the oxidation of capric acid.

The difficulties encountered in the calcula­

tion of lag periods have previously been discussed; the question of the significance of short lag periods is still unanswered.

The periods in the oxidation of the alpha-

beta unsaturated and the beta hydroxy acids were 2 6 and 40 minutes respectively; as a result of growth on caprate these periods were reduced to 8 minutes.

It would seem

permissible to assume that this represents adaptation to these acids and that the observed lag periods are due to technical difficulties both in the conduct of the ex­ periments and in the mechanics of calculating the lag. This leads to the conclusion that all three derivatives of capric acid are intermediates in its oxidation by Alphin cells. The relationship between adaptation to caprate derivatives and the oxidation of other derivatives.

Glu­

cose-grown cells were specifically adapted to the three caprate derivatives by adding 0 . 6 ym of substrate to each

106

ml of cell suspension and shaking in Warburg Vessels. After the added substrate was utilized (as indicated by return to autorespiratory oxygen uptake) the test sub­ strates were poured from the side-arms. Figures 9> 10, and 11 show that exposure of glucosegrown cells to any of the three caprate derivatives caused a shortening of the lag periods in the oxidation of the other two compounds.

Organisms specifically adapted to

alpha-beta unsaturated capric acid show no lag in the ox­ idation of the beta hydroxy or the keto acids.

Beta hydroxy

caprate exposed cells showed no lag in metabolizing the keto acids.

Beta hydroxy caprate exposed cells showed no lag in

metabolizing the keto and the unsaturated compounds.

Cells

adapted to the keto acid had a 9 minute lag in the oxida­ tion of the hydroxy acid and an 8 minute lag in the oxida­ tion of the unsaturated derivative of caprate. Adaptation of cells to the oxidation of the hydroxy and keto derivatives as a result of exposure to the un­ saturated acid indicates that the keto and hydroxy acids are intermediates in the oxidation of the alpha-beta un­ saturated compound.

The simultaneous adaptation of beta

hydroxy caprate-exposed cells to the keto derivative supports this conclusion.

Although it is inconceivable

that alpha-beta unsaturated caprate is an intermediate in the oxidation of the beta hydroxy acid, adaptation to the

107 hydroxy derivative simultaneously adapted organisms to the unsaturated acid.

This suggests a close relationship

between the two compounds.

Chemically the hydroxy acid

differs from the unsaturated derivative in that the former is fully saturated with a molecule of water.

It may be

that in the cell the unsaturated acid is in equilibrium with and rapidly converted to the hydroxy acid through the action of a reversibly acting enzyme; similar enzymes are known to be involved in the reversible conversion of fumarie acid to malic acid and in the conversion of cis aconitic acid to citric acid.

If an enzyme exists in the

cell for the conversion of alpha-beta unsaturated fatty acid to beta hydroxy fatty acid, then one would expect that adaptation to one of the compounds would result in simultaneous adaptation to the other. The relatively short lag periods noted when beta keto capric acid exposed cells were allowed to oxidize the hydroxy and the unsaturated acids are probably indicative of simultaneous adaptation to the oxidation of these deriva­ tives as a result of exposure to the keto acid.

The results

reported in the next section support this conclusion. The effect of adaptation to caprate derivatives on the oxidation of capric, caprylic, and undecylic acids.

In

order to determine the effect of adaptation to caprate derivatives on the oxidation of the normal saturated acids,

108

glucose-grown cells were specifically adapted to the three caprate derivatives and then allowed to oxidize caprate, caprylate, and undecylate. Table V.

The results are shown in

Adaptation of cells to any one of the three com­

pounds produced organisms which were adapted to oxidation of the C3 ,

an(*

acids.

As in the previous sec­

tions, there were instances where the lag periods were not completely eliminated, but the great differences between the calculated lag periods for exposed and unexposed cells allows the conclusion that exposure to any of these three compounds adapts the cells to the three normal fatty acids tested.

DISCUSSION Although aspects of the data already discussed (i.e., the oxidation of caprate in the presence of DNP by glucosegrown cells) makes it impossible to conclude definitely that Serratia marcescens (Alphin) attacks fatty acids by means of the "classical" type of adaptive enzymes, it is certain that exposure to or growth on fatty acids or their derivatives profoundly affects the metabolism of this organ­ ism with respect to this group of compounds.

The basic

theory behind the concept of simultaneous adaptation (Stanier, 19^7) or 11simultaneous activation" (Ajl, 1950) will still apply, even though qualitative differences in the properties of the enzymes exist.

109

TABLE V LAG PERIODS IN THE OXIDATION OP CAPRIC, UNDECYLIC, AND CAPRYLIC ACIDS IN RELATION TO ADAPTATION TO CAPRIC ACID DERIVATIVES ^ ^ Oxidation of

Lag period in minutes after specific adaptation alpha-beta beta hyaroxy beta keto Unadapted unsaturated capric acid capric acid capric acid

caprylic

5

3

6

25

capric

0

0

7

30

undecylie

2

6

4

32

Specific adaptation accomplished by exposing Serratia (Alphin) cells to 0.6 micromole of derivative per ml of cell suspension. Unadapted cells were unexposed. All cells were glucose-grown. Flask concentrations of fatty acids:

0 .0 0 0 3 M.

Experiments conducted in air atmosphere with 10 per cent KOH to absorb carbon dioxide. Temperature--30°C. Lag periods estimated by extension of steepest part of curve to time axis.

110

The data indicate that the alpha-beta unsaturat­ ed, beta hydroxy, and beta keto derivatives of capric acid are intermediates in the oxidation of caprate. The evidence for this is conclusive, since adaptation of cells to capric acid simultaneously adapts the organ­ isms to the oxidation of the three caprate derivatives. Chemical logic would dictate that the oxidative path­ way should involve a preliminary oxidation of capric acid to its unsaturated derivative and that this should be followed by formation of the hydroxy and then the keto acid.

An examination of the formulae of these compounds

indicates that an alpha-beta dehydrogenation should be the primary attack on capric acid and also on its hydroxy derivative.

Neither the keto acid nor the unsaturated

acid can undergo an alpha-beta dehydrogenation, yet, adaptation to either of these compounds simultaneously adapts Serratia cells to the saturated and hydroxy acids. The adaptation to the hydroxy acid is expected, since this compound should be an intermediate in the oxidation of alpha-beta unsaturated capric acid; but neither the un­ saturated acid, the hydroxy acid, nor capric acid can be intermediates in the oxidation of beta keto capric acid. To complicate the picture, adaptation of cells to any of these four compounds simultaneously adapts the organisms to the oxidation of undecylic acid, an eleven carbon com­ pound.

Furthermore, previous studies showed that

Ill adaptation to any of the acids oxidized by the Alphin strain simultaneously adapted the cells to the oxida­ tion of all the other acids.

These facts suggest that

a single enzyme system catalyzes the oxidation of all the acids oxidized by the Serratia cells. might be postulated:

Two mechanisms

Scheme I— Beta Oxidation and Scheme

II— Multiple Alternate Oxidation as shown on the follow­ ing page.

SCHEME I— BETA OXIDATION C-C-C-C-C-C-C-C-C-COOH capric acid C-C-C-C-C-C-C-C=C-COOH

* C-C-C-C-C-C-C-C=C-C00H (1) alpha beta unsaturated capric acid . C-C-C-^5^C-C-CH0H-C-C00H ( (2) 2) beta hydroxy capric acid w TT^C-C-C-C-C-CO-C-COOH (3) 2H beta keto capric acid TOTC-C-C-C-C-COOH / C2 fragment * caprylic acid (4) W C-C-C-C-C-COOH / C 2 fragment (2) (3) (4) caproic acid C-C-C-COOH / C2 fragment (2 )(3 ) (4 ) butyric acid 2 C2 fragments (2) (3) (4)

- 2H

r

C-C-C-C-C-C-C-CHOH-C-COOH

C-C-C-C-C-C-C-CO-C-COOH

T

C-C-C-C-C-C-C-COOH

reactions(1) C-C-C-C-C-COOH

reactions (1 )

C-C-C-COOH reactions (l)

SCHEME II— MULTIPLE ALTERNATE OXIDATION C-C-C-C-C-C-C-C-C-COOH capric acid C-C-C-C-C-C-C-CO-C-COOH

reactions (l) (2 ) (3 ) _

_

C-C-C-C-C-C=C-CO-C-COOH

C-C-C-C-C-C-C-CO-C-COOH * beta keto capric acid ^ CTcTc-C-C-C=C-C0-C-C00H

(1 )

^ C-C-C-C-C-CHOH-C-CO-C-COOH (2)

C-C-C-C-C-CHOH-C-CO-C-COOH

C-C-C-C-C-CO-C-CO-C-COOH

(3)

- 2H C-C-C-C-C-CO-C-CO-C-COOH

C-C-C-CO-C-CO-C-CO-C-COOH reactions (1 ) (2 ) (3 ) C-CO-C-CO-C-CO-C-CO-CrCOOH

C-C-C-CO-C-CO-C-CO-C-COOH reactions (l) (2 ) (3 ) C-CO-C-CO-C-CO-C-CO-C-COOH

^ 5 C2 fragments (4a) / 4 H20 112

113 The initial steps are the same in both schemes. They involve (l) the conversion of saturated acid to alphabeta unsaturated acid by removal of two hydrogen atoms, (2 ) the conversion of unsaturated acid to beta hydroxy acid by addition of water, and (3 ) the conversion of hydroxy acid to keto acid by removal of two more hydrogen atoms. Since reactions 1 and 3 involve dehydrogenation at the alpha-beta position, the possibility exists that a single enzyme may catalyze both reactions. Since both the saturat­ ed acid and its hydroxy derivative are oxidized by glucosegrown Serratia cells only after a lag period, it follows that reactions 1 and 3 are catalyzed by adaptive enzymes. Reaction 2 involves addition of water at the alpha-beta position, and hence the enzyme active in this step must be different from those involved in steps 1 and 3»

This

reaction is analogous to the reversible conversion of fumaric to malic acid which occurs in the tricarboxylic acid cycle and is catalyzed by the enzyme fumarase.

The data

do not indicate whether the enzyme involved in reaction 2 is constitutive or adaptive, since conversion of unsaturat­ ed acid to hydroxy acid produces a compound which itself is attacked by adaptive enzymes.

These three reactions

and the enzymes catalyzing them must be involved in the oxidation of all the fatty acids oxidized by the Serratia cells, since adaptation to any of these acids simultaneously

114 adapts the cells to the oxidation of all the other acids. The peculiar position of acetic acid will be discussed later. The two schemes differ in the mechanism of keto acid oxidation.

The first mechanism visualizes classical

beta oxidation occurring.

Accordingly, the keto acid

undergoes hydrolytic cleavage with the formation of a twocarbon fragment and a fatty acid with two less carbon atoms. Since the lower acid should be oxidized through the same pathway as the higher homologue, reactions 1 , 2 , and 3 repeat themselves and are catalyzed by the same enzymes that were involved in the oxidation of the higher acid. Reaction 4, the cleavage of beta-keto acid, may be catalyzed by either an adaptive or a constitutive enzyme.

The data do

not indicate which type is involved, since the cleavage of keto acid produces a compound which is attacked through adaptative enzymes.

This mechanism would explain the adap­

tation of cells to the oxidation of capric acid and its hydroxy and unsaturated derivatives after exposure to beta keto acid, since adaptation to the beta keto compound simul­ taneously adapts the cells to reactions 1 , 2 , and 3 in the oxidation of caprylate.

The enzymes catalyzing these re­

actions are the same as those catalyzing the first three steps in caprate oxidation. According to Scheme II, the keto acid formed as a

115 result of reactions 1 , 2 , and 3 does not cleave, but a second oxidation occurs at the delta carbon atom.

As a

result a second keto group is formed; subsequently, simi­ lar oxidations occur at alternate carbon atoms on the fatty acid with the eventual formation of a polyketo acid.

According to this scheme, reactions 1 , 2, and 3

repeat themselves, i.e. the enzymes Involved in the forma­ tion of the delta keto group are the same as those catalyz­ ing the formation of beta keto acid.

The results from ex­

periments with simultaneous adaptation would indicate that these enzymes are involved in the formation of polyketo acids from all the oxidizable acids (except acetic).

Re­

action 4a, the cleavage of polyketo acids to give twocarbon fragments, may or may not be catalyzed by an adap­ tive enzyme. The question arises as to the significance of acetic acid in the oxidation of higher acids by Serratia cells. When glucose-grown Serratia cells are exposed to acetate, they become adapted not only to acetate oxidation but also to the oxidation of higher acids.

It has been postulated

that higher acids are oxidized to beta keto acids; acetic acid, being a two-carbon compound, cannot be metabolized in this manner.

This suggests that acetate is an inter­

mediate in the oxidation of both odd and even-chain acids and indicates that the reactions involved in acetate

116

formation are reversible.

Rittenberg and Bloch (1945)

have shown that in animal tissue higher even chain acids are synthesized from acetate.

The results would indicate

that a similar mechanism occurs in Serratla. The synthet­ ic process must be the reverse of the oxidation reaction. Both schemes suggested indicate that two-carbon fragments arise from the cleavage of keto acids.

The re­

sults from experiments with DNP indicated that acetate is not a direct intermediate in the oxidation of higher acids. It was suggested, however, that an "active” form of acetate may be an intermediate.

It follows that this two-carbon

fragment, related to acetate, must also be active in the reverse reaction, i.e. the synthesis of higher acids from acetate. The experimental data does not allow an unequivocal choice between the two suggested mechanisms.

The failure

to demonstrate oxidation of propionic, butyric, and valeric acids by Serratia cells would appear to rule out beta oxi­ dation in its classical conception, since butyric acid should be an intermediate in the oxidation of higher even chain acids.

Since the higher acids are completely oxidiz­

ed to carbon dioxide and water, these substances do not accumulate. 1.

Three possibilities suggest themselves: The Serratia cells are impermeable to butyric,

propionic, and valeric/acids, but these substances can be

117 oxidized when formed in the cell as the result of the oxi­ dation of higher acids. 2.

These lower acids cannot be oxidized as such,

but during the oxidation of higher acids, "active" three, four, and five-carbon compounds are formed, and these "ac­ tive" substrates undergo oxidation. 3.

Propionic, butyric, and valeric acids are per­

meable to the cells but are not oxidized. The first postulate, if correct, would mean that beta oxidation in its classical conception is the mode of fatty acid oxidation in Serratia.

In this case, cell free

enzyme preparations should as readily oxidize the C3 , C^, and C5 acids as they do the higher acids.

If "activated"

forms of C 3 , C4 , and C 5 compounds are intermediates, then the essential features of beta oxidation are retained, but the chemical nature of the intermediate compounds is some­ what different than generally supposed.

The work with

DNP indicated, it will be recalled, that neither caprylic nor caproic acids are direct intermediates in the oxida­ tion of caprate and similarly that heptylate is not a direct intermediate in the oxidation of pelargonic acid. There is good evidence, then, that if beta oxidation oc­ curs the intermediates are not the "normal" form of the saturated acids, but direct evidence is still lacking for the occurrence of "active" forms of the C 3 ,

and

acids

118 as intermediates in the oxidation of higher acids.

If

oxidation were to occur by the second scheme, i.e. mul­ tiple alternate oxidation, then it would possible for high­ er acids to be oxidized without the formation of propionic, butyric, or valeric acid.

Such a mechanism would lead to

the formation of odd chain dicarboxylic acids during the oxidation of odd chain acids.

It appears that these sub­

stances might be oxidized through a terminal decarboxyla­ tion of one of the acid groups with the formation of an even chain monocarboxylic acid. It would seem that work with cell-free enzyme systems would be the most promising approach to the problem of determining the relationship of the C^, C^, and to the oxidation of higher acids.

acids

An understanding of

this relationship would lead to clarification of the basic mechanism whereby saturated acids are oxidized by the Serratia cells.

SUMMARY Q-lpha-beta unsaturated, beta hydroxy, and beta keto derivatives of capric acid were oxidized by glucosegrown Serratia (Alphin) cells after lag periods.

Cells

harvested from capric acid-mineral salts medium showed no lag periods in the oxidation of the three derivatives of capric acid.

Cells adapted to the oxidation of any one of

119 the three derivatives were also adapted to the oxidation of the other two compounds and also to the oxidation of capric, caprylic, and undecylic acids. The results suggested that a single enzyme system catalyzes the oxidation of all the fatty acids known to be oxidized by the Serratia cells.

The preliminary steps

appear to involve (1 ) alpha-beta dehydrogenation of saturat­ ed acid with the formation of an alpha-beta unsaturated fatty acid, (2 ) addition of water at the double bonds with the production of a beta hydroxy fatty acid, and (3 ) alphabeta dehydrogenation of the hydroxy acid with the formation of beta keto fatty acid.

Two mechanisms for beta keto acid

oxidation were proposed, i.e. classical beta oxidation and multiple alternate oxidation.

The data are insufficient to

allow an unequivocal choice between the two pathways. The role of acetate In the metabolism of higher acids was discussed.

Although the data indicate that acetate Is

not a direct intermediate in the oxidation of higher acids, a two-carbon fragment readily formed from acetate must be an intermediate in the oxidation of both odd and even chain acids.

CHAPTER VI THE RELATIONSHIP BETWEEN THE CITRIC ACID CYCLE AND THE OXIDATION OF FATTY ACIDS BY SERRATIA MARCESCENS Numerous investigators have shown a relationship between the citric acid cycle and the oxidation of fatty acids in animal tissues.

Compounds in this cycle appar­

ently have two distinct functions with respect to the oxida­ tion of fatty acids:

(l) Grafflin and Green (19^8) and

Knox et al. (19^8) have shown that the oxidation of a small amount of a citric acid cycle compound is an obligatory "sparking” reaction in the oxidation of fatty acids by the cyclophorase system prepared from rabbit kidney.

All at­

tempts to demonstrate fatty acid oxidation by the cyclo­ phorase system without cooxidation of tricarboxylic acid cycle compounds have failed.

(2)

When fatty acids are

oxidized to completion, the terminal oxidation of these substances involves a coupling reaction with oxalacetate to form citric acid or a closely related compound.

The con­

densation involves acetate or acetoacetate, substances pro­ duced in the primary oxidation of fatty acids (Green, 1 9 ^8 ). To a lesser extent, higher beta-keto fatty acids may con­ dense directly with oxalacetate to form a labile compound which yields citric acid and a saturated fatty acid with two less carbon atoms than were contained in the keto acid

121

participating in the condensation (Breusch, 1948).

The

sparking reaction initiating fatty acid oxidation and the condensation reaction involved in the terminal oxidation of fatty acid derivatives are independant processes, even though tricarboxylic acid cycle compounds are active in both reactions. It seemed important to determine the relationship between the citric acid cycle and the oxidation of fatty acids by Serratia: 1.

Evidence from the previous studies indicated that

the two, eight, nine, and ten-carbon acids are oxidized to completion by these bacteria, and there were indications that the six and seven-carbon acids are also completely oxidized.

The question thus arises as to the mode of ter­

minal oxidation of these compounds. 2.

Previous work suggested the possibility that the

lag periods in the oxidation of fatty acids by glucosegrown cells might be due to a time necessary for the accumu­ lation of some compound necessary for the oxidation of fat­ ty acids.

It was postualted that such a compound might be

a member of the citric acid cycle.

EXPERIMENTAL The methods employed were the same as described in previous chapters.

All suspensions used were adjusted to a

122

standard turbidity measured on the Klett-Summerson ap­ paratus*

Cells were harvested from glucose or capric

acid medium and treated in the manner described in prev­ ious sections.

One experiment was conducted in which cells

grown on a succinate medium were used.

In this case the

mineral salts medium contained 0 . 0 1 M succinic acid as the sole source of carbon; these cells were grown at room temperature and harvested at 40 hours.

Growth at 37°C.

was very slow when succinate was the sole source of carbon. The oxidation of citric acid cycle compounds by Serratia.

Glucose-grown cells were tested for ability to

oxidize succinic, malic, citric, and oxalacetic acids.

Ci­

tric acid was not metabolized but had no inhibitory effect on endogenous respiration.

Oxygen uptake in the presence

of the other three compounds was about 5 0 per cent of the amount required for complete oxidation.

No attempt was

made to block assimilation with DNP, but if one assumes that oxidative assimilation is as great as that found when fatty acids are oxidized, then it follows that the oxida­ tion of malate, succinate, and oxalacetate is complete. The effect of prior oxidation of succinate and malate on the oxidation of capric acid. Experiments were conducted in which glucose-grown cells were exposed to malate and succinate in Warburg vessels.

After added sub­

strate had been oxidized, capric acid was poured from the

123 side-arm, and the oxygen uptake by the exposed cells was measured.

This prior oxidation of citric acid cycle

compounds had no effect on the pattern of fatty acid oxida­ tion by glucose-grown cells; capric acid was oxidized after a lag period of the same length observed with unexposed cells.

Cells grown on succinic acid medium also oxidized

caprate with an initial lag period characteristic of glucose-grown cells. The effect of cooxidation of citric acid cycle com­ pounds .

Experiments were conducted in which Serratia

cells were tested for caprate oxidation in the presence of citric acid cycle compounds. 0 .0 0 0 3 M concentration;

Capric acid was used in

in most instances, the tricarboxyl­

ic acid cycle compounds were added in a concentration equimolar with the fatty acid, but in certain experiments higher concentrations were used.

Oxygen uptake was also

measured in flasks containing capric acid and citric acid cycle compounds separately.

The results for malate.are

plotted in Figure 12 and tabulated in Table VI.

The data

for citrate, succinate, and oxalacetate are shown in Table VII.

The figures presented in the two tables repre­

sent averages obtained from duplicate (and 7*'sometimes triplicate) flasks. Figure 12 shows that capric acid was oxidized by glucose-grown cells after a lag period of approximately

124 i»o-

1 70* ■

1 40 - -

IIO - -

7 0 --

30-

20-

1060 |

80

IO O T IM E

120 IN

140

160

I BO

to o

120

£40

M IN U T E S

M/20 phosphate buffer, pH 7*0, 30°C., 0.1 ml 10 per cent KOH in center well. Flask concentrations: malate as indicated in figure; caprate, 0.0003 M. Where both sub­ strates were present, they were added simultaneously from the side-arm. FIGURE 12 THE OXIDATION OF CAPRATE IN THE PRESENCE AND ABSENCE OF MALATE

125 TABLE VI CAPRIC ACID OXIDATION IN RELATION TO THE SIMULTANEOUS OXIDATION OF MALATE

Substrate

0 . 0 0 0 3 M. malate (l)

0.0003 M. caprate (2) Total (1) (2) malate /.caprate

Cells

glucose grown

Total Oxygen uptake In microliters at the end of * 20 40 . 60 minutes minutes minutes 6 2 8 15

11 19

16

28

37 53 44

30

0.0015 M. malate (l) 0.0003 M. caprate (2) Total (1) (2) malate / caprate

glucose grown

10 2 12 18

34 19 53 48

59 37 96 74

0 . 0 0 1 5 M. malate (1)

caprate grown

16

51 6l 112 73

77 83

0.0003 M. caprate (2) Total (1) (2) malate / caprate

24 40 29

160

114 ‘

Experiments conducted in air atmosphere with 0.1 ml 10 per cent KOH in center well. Temperature 30°C. In flasks containing malate / caprate# both substrates were added simultaneously from the side-arm af£er-~the usual 15 minute equilibration period. ' *