THE .J~URN~L OF RIOLOOICAL CHEMISTRY Vol. 246, No. 9, Issue of May 10, pp. 2765-2771, 1971

P&ted in U.S.A.

Biohydrogenation of Unsaturated Fatty Acids

V. STEREOSPECIFICITY OF PROTON ADDITION AND MECHANISM OF ACTION OF LINOLEIC ACID A12-cis,A11-trans-ISOMERASE FROM BUTYRIVIBRIO FIBRISOLVENP

(Received for publication, October 28, 1970)

CAROL R. KEPLER, W. P. TUCKER, AND S. B. TOVE

From the Departments of Biochemistry and Chemistry, North Carolina State University, Raleigh, North Carolina 27607

SUMMARY

The specificity of linoleic acid isomerase from Bufyriuibrio jibrisolvens for cis-9,cis-1%dienoic fatty acids with an o chain length varying from 4 to 8 carbons has been examined. The enzyme was found to be highly specific for a straight chain fatty acid bearing an w chain length of 6 carbon atoms.

Stereospecific addition of hydrogen to carbon atom 13 of linoleic acid in the D configuration was demonstrated. It was deduced that the substrate was bound to the enzyme in the form of a loop and that the mechanism of isomerization involves either the protonation of an enzyme-bound carbanion or a concerted reaction. A tentative model with the carboxyl oxygens of the substrate participating in the isomerization reaction is proposed.

Linoleic acid A12-cis, A”-trans.isomerase’ catalyzes the first reaction of the biohydrogenation of linoleic acid to truns-ll- octadecenoic acid (l-3). Previous studies of the substrate specificity of linoleic acid isomerase have delineated a strict requirement for a free carboxyl group and a cis-9, cis-12-diene system. The specificity studies have now been extended to an examination of the effect on isomerization of varying the size of the w chain of cis-9, cis-12-diene acids. We have shown that the hydrogen added to carbon atom 13 of cis-9, trans-1 l-octa- decadienoic acid in the isomerization reaction rapidly equili-

* Contribution from the Departments of Biochemistry and Chemistry, School of Agriculture and Life Sciences, and School of Physical and Mathematical Sciences. Paper 3321 of the Journal Series of the North Carolina State University Agricultural Experi- ment Station, Raleigh, North Carolina. This work was supported in part by Public Health Service Research Grant AM-02483 from the National Institute of Arthritis and Metabolic Diseases. The high and low resolution mass spectrometry wasd one at the Re- search Triangle Institute Center for Mass Snectrometrv. Research Triangle Park, North Carolina, under Grant PR 336’from the Biotechnology Resources Branch of the National Institutes of Health.

1 The name of this enzyme was originally designated as linoleate A1’%s,A1%“ans-isomerase (2, 4). However, since the active form of the substrate appears to be the undissociated acid, we now propose the name linoleic acid A12-~is,A11-tru~.s-isomerase.

brates with water, suggesting a protic mechanism (2). The results of an investigation of the stereospecificity of the protona- tion with 12, 13-3H-linoleic acid are presented in this paper. Kinetic analysis of the isomerization of deuterated substrates provided further insight into the reaction mechanism.

EXPERIMENTAL PROCEDURE

Substrates and Reagents

The commonly occurring fatty acids were purchased from Hormel. Deuterium oxide was obtained from Stohler Isotope Chemicals. Perdeuterolinoleic acid was a gift of James A. McCloskey of Baylor University College of Medicine. When necessary, purification and saponification of the methyl esters of fatty acids were carried out as previously described (4).

Methyl II, 11-Dideuterolinoleate and Methyl 11 Jl-Dideu.terooleate

The synthesis of methyl 11 , 11-dideuterolinoleate was achieved by the condensation of 9-decynoic acid with 1 ,l-dideutero-l- bromo-2-octyne, followed by catalytic reduction of the two acetylenic bonds. The details of this procedure and criteria of purity are described elsewhere (5). The synthesis of the 11, ll- dideuterooleate, also detailed elsewhere (6), involved a Wittig condensation of 2,2-dideuterononanal and g-carbomethyoxy- nonyltriphenylphosphonium bromide. The dideuteroaldehyde was prepared by pyridine-catalyzed exchange of the a-hydrogens of nonanal with deuterium oxide.

Methyl cis-9,cis-1%Dienoate Homologues of Methyl Lirwleate

The synthesis of methyl cis-9, cis-12-heptadecadienoate has been described (4). Each of the methyl cis-9, cis-12-dienoates (methyl hexadecadienoate, methyl 16-methylheptadecadienoate, methyl nonadecadienoate, and methyl eicosadienoate) was synthesized by condensing the appropriate I-bromo ,2-yne derivative with 9-decynoic acid as described for methyl cis-9, cis- 12-heptadecadienoate (4). All of the acetylenic bromides except the branched bromide were prepared from the appropriate acety- lenic alcohols (7) purchased from Farchan Research Laboratories, Willoughby, Ohio. The branched alkyne, 1-bromo-6-methyl-2- heptyne, was synthesized by reacting 5-methyl-1-hexyne with ethyl magnesium chloride (Fisher) and paraformaldehyde ac- cording to the procedure of Taylor and Strong (7). The alcohol derivative was purified by vacuum distillation, b.p. 61-80” (2

2765

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

2766 Biohydrogenation of Unsaturated Fatty Acids. V Vol. 246, No. 9

mm), and converted to the bromo derivative with phosphorous tribromide in pyridine and ether (7), yielding an oil that was isolated by vacuum distillation, b.p. 58-60” (2 mm).

Following hydrogenation (8), each ester was isolated by a combination of column chromatography on Florisil and on silver nitrate-impregnated silicic acid (9) and preparative gas- liquid chromatography. Characterization and purity were established by high and low resolution mass spectrometry, gas- liquid chromatography, and thin layer chromatography. The position of the double bonds was established by reductive ozon- olysis (1). Each ester produced an aldehydo-ester fragment which when analyzed by gas-liquid chromatography, emerged with the same retention time as methyl azelaldehyde. Ultra- violet and infrared spectroscopy confirmed the absence of any conjugated or tram double bonds.

Methyl Hexadecadienoate-

C&LOOS Nuclidic mass calculated: 266.2246 Found : 266.2241

Methyl 26-Methylheptadecadienoate-

Ct9&02

Nuclidic mass calculated : 294.2559 Found : 294.2559

iMethyl Nonadecadienoate-

Cz&~Os Nuclidic mass calculated: 308.2715 Found : 308.2709

Methyl Eicosadienoate-

CzlHasOz

Nuclidic mass calculated: 322.2872 Found : 322.2881

od&icinoleic Acid

A procedure similar to that of Schroepfer and Bloch (10) was used to prepare the tosyl derivative of methyl n-ricinoleate. Heating the tosyl derivative at 60” with anhydrous sodium ace- tate, acetic acid, and benzene gave the acetoxy derivative which was isolated and purified by preparative gas-liquid chromatog- raphy. This product proved to be optically inactive. Authen- tic methyl n-acetoxyricinoleate exhibited [a] t5 = 21.8” (acetone) and was chromatographically identical with the racemic product. Saponification to ricinoleic acid was carried out in 0.5 M potas- sium hydroxide in 50% ethanol under nitrogen.

Methods

Culture of the organism, preparation of the enzyme, and spec- trophotometric assays were carried out as previously described (11). Kinetic data were analyzed by computer with a slightly modified program of Cleland (12). Protein was determined by reaction with phenol reagent (13).

Determination of Xtereospeci$city of Hydrogen Addition

The 12, 13-3H-cis-9, cis-12-octadecadienoic acid2 was prepared by catalytic reduction (8) of cis-9-octadecen-12-ynoic acid kindly furnished by R. L. Anderson of Proctor and Gamble. Prepara-

z Hydrogenation of 17 mg of acid (60 pmoles) with 3 Ci (about 4Opmoles) of tritium gas was performed by New England Nuclear.

tive thin layer chromatography on silica gel impregnated with 25% silver nitrate with a solvent mixture of pentane-ether (9 : 1) was used to purify the tritiated linoleate after methylation with diazomethane. After saponification, 10 PC1 of the ditritiolinoleic acid and 0.1 mg of carrier were mixed with 1 PC1 of l-14C-linoleic acid (New England Nuclear). The doubly labeled linoleic acid was incubated at 30” for 3 hours with 25 mg of isomerase protein in 2 ml of 0.1 M phosphate buffer, pH 7.0, containing 4 mg of bovine serum albumin (Fraction V), which had been freed of fatty acids by charcoal treatment (14). Following extraction by the procedure of Dole (15), the fatty acids were treated with diazomethane and the methyl cis-9, trans-1 l-octa- decadienoate was isolated by preparative silver nitrate thin layer chromatography. The conjugated ester was saponified and the acid reduced by vigorous shaking with hydrazine hydrate (16). After treatment again with diazomethane, the doubly labeled methyl stearate was isolated by silver nitrate thin layer chroma- tography and saponified.

The doubly labeled stearic acid was then incubated with a suspension of ChZorella vuZgarisa as described by Harris, Harris, and James (17). After a 21-hour incubation at 20”, the cell suspension was extracted with chloroform-methanol (18) and the lipids were saponified. Following methylation, the linoleate and unreacted stearate were isolated by thin layer chromatography on silicic acid impregnated with silver nitrate. Final purification of each ester was achieved by vacuum distillation. The activity of each isotope was measured in a liquid scintillation spectrometer and was corrected for double label as previously described (19).

Incorporation of Deuterium into Substrate and Product from Deu- terium Oxide

Each incubation was carried out in a cuvette equipped with a large volume adapter and followed spectrophotometrically (11) to determine the time course of the reaction. Identical reac- tions consisting of linoleic acid (0.48 mg) and 0.2 ml of enzyme (11 mg of protein) in 24 ml of 0.1 M phosphate buffer, pH 7.0, in deuterium oxide were run consecutively for different time intervals. Each reaction was quenched by pouring the entire incubation mixture into a solution of isopropanol, isooctane, and 1 N sulfuric acid (40 : 10 : 1). The fatty acids were isolated (15) and treated with diazomethane. Methyl linoleate and methyl cis-9, trans-1 l-octadecadienoate were then analyzed for deu- terium content by a mass spectrometer (LKB) equipped with a gas-liquid chromatographic inlet system.

Exchange of Deuterium in 11 ,il-Dideuterooleic Acid

The 11,l I-dideuterooleic acid (0.8 mg) was incubated with 17 mg of isomerase protein in 60 ml of 0.1 M phosphate, pH 7.0, at 37” for 3.5 hours. Following extraction (15) and methylation with diazomethane, the methyl oleate was purified by column chromatography on silver nitrate-impregnated silicic acid and was analyzed by mass spectrometry. A duplicate flask not in- cubated served as a control.

RESULTS

Effect of Chain Length of AsJ2-Fatty Acids-Previous studies of linoleic acid isomerase demonstrated an absolute specificity for a substrate that contained a free carboxyl group and a cis-9, cis-

3 Kindly furnished by A. T. James of Unilever Research Labo- ratory, Colworth House, Sharnbrook, Bedford, England.

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Issue of May 10, 1971 C. R. Kepler, W. P. Tucker, ancl X. B. Tove 2767

TABLE I TABLE II Efect of chain length on linoleic acid isomerase

The incubation mixture for each assay contained 90 rnM potas- sium phosphate buffer (pH 7.0), 10% 1,3-propanediol, and 15 PM substrate acid in a final volume of 3.0 ml. The reaction was con- ducted at 35” and was initiated by the addition of 0.7 mg of enzyme protein. Each value is the a,verage of four replicat,e assays.

cis-9,&s-12.Fatty acid Activity

nmoles/min/mg protein

1632a 2.1 17:2 7.3 l&Methyl 17:2 3.1 18:2 13.0 19:2 2.0 20:2 0

Kinetic constants of linoleic acid isomerase Each cuvette contained fatty acid (5 to 30 FM) in 10% 1,3-

propanediol in 0.1 M phosphate buffer (pH 7.0) in a total volume of 3.0 ml. The reaction was initiated by the addition of 0.3 to 0.5 mg of enzyme protein. The kinetic constants were calculated by computer, the data for linoleic acid representing the average of 12 determinations, each of which comprised an average of five repli- cations, and the data for the other acids representing the average of two determinations, each of which is also the average of five replications.

Substrate acid

0 The number preceding the colon indicates the chain length of the fatty acid; the number following the colon indicates the number of double bonds.

12-diene system (4). In these experiments, the only cis-9 ,cz’s- 12-dienoic acid examined other than linoleic acid was heptadeca- dienoic acid, which was isomerized at half of the rate of linoleic acid.

Linoleic Ag~r2*r~-Octadecatrienoic. . 18.4 f 3.7 56.8 h 6.6 A6ag,12-Octadecatrienoic. 10.5 It 1.3 13.9 Zk 1.9 ll,ll-Dideuterolinoleic . 7.4 f 1.5 14.1 f 0.9 Perdeuterolinoleic . . 9.4 rt 1.4 13.2 zk 0.7

1 Q The maximum velocities have been normalized to correct for

differences in activity and amount of protein of the enzyme prepa- rations.

b Standard deviation.

In order to investigate further the effect of chain length of cis-9, cis-%dienes, methyl hexadecadienoate, nonadecadienoate, eicosadienoate, and 16-methylheptadecadienoate were synthe- sized. When the individual acids of this series were assayed at a concentration of 1.5 x lop5 M, marked specificity was observed for linoleic acid (n-6)4 (Table I). As the w chain length increased from 4 to 6 carbons, there was a progressive increase in isomeriza- tion. Ueyond an w chain length of 6 carbons, isomerase activity decreased rapidly, with cis-9, cis-12-eicosadienoic acid (n-8) inac- tive as a substrate. The number of carbons in the w chain is not the sole determinant of fit to the hydrophobic pocket since the rate of isomerization of the 16.methylheptadecadienoic acid was less than either the 17.carbon or 1%carbon straight chain dienoic acids. Even though the 20-carbon homologue did not function as a substrate, the presence of 24 pM cis-9,&s-12-eicosadienoic acid resulted in 52% inhibition of the isomerization of 6 pM

linoleic acid.

H-6-H H-6-H H-6-T

H-6-H T-d-H

T-+-H H-6-T

H-&H

T-4-H T-d-H

H-#-T H-+-T

3

I H-&H H-l:

Kinetic Constants of Octadecattienoic Acids-In addition to linoleic acid, two other naturally occurring 18.carbon unsatu- rated acids, cis-9, cis-12, cis-15.octadecatrienoic acid (cY-linolenic) and cis-6, cis-9, cis-12.octadecatrienoic acid (y-linolenic), are isomerized by linoleic acid isomerase (4). In previous studies it was found that the apparent K, for AgJ2J5-octadecatrienoic acid was about double that of linoleic acid (a), yet it had been con- sistently observed that linolenic acid was isomerized more rapidly than linoleic acid. These observations are confirmed by the data of Table II. Thus, the expected decrease in relative velocity for linolenic acid occasioned by the higher Km is more than offset by the increased rate of catalysis.

FIG. 1. Scheme for determination of the stereospecificity of hydrogen addition to carbon 13 of linoleic acid by linoleic acid isomerase. Each structure represents carbon atoms 11 to 13 of linoleic acid. The sequence of reactions is (1) 12,Wditritiolino- leic acid is isomerized by linoleic acid isomerase to form the cis- S,trans-11-octadecadienoic acid bearing a hydrogen of either D or L configuration, (2) hydrogenation of the specifically labeled conjugated acid to stearic acid, followed by (3) stereospecific desaturation to linoleic acid by Chlorella vulgaris which yields a product that retains 75y0 of the tritium in the case of D addition and 25% of the tritium in the case of L addition.

In contrast, insertion of a double bond between the AgJ2-diene system and the carboxyl group, as in cis-6, cis-9, cis-Uoctadeca- trienoic acid, causes no change in the K, but markedly reduces the catalytic activity (Table II).

4 In accordance with the newly revised lipid nomenclature (J. Biol. Chem., 246, 1511 (1970))) the w chain length is indicated by (n-x) where n corresponds to the number of carbon atoms in the chain and 2 corresponds to the o chain length.

XtereospecQicity of Hydrogen Addition to Carbon 13-A study of the stereospecific addition of a hydrogen to carbon 13 during the isomerization of linoleic acid was conducted according to the following procedure (Fig. 1). A mixture of 12,13-ditritiolinoleic acid and l-i4C-linoleic acid was incubated with linoleic acid isomerase. The cis-9, trans-11-octadecadienoic acid produced was isolated and converted to stearic acid by the addition of hydrazine. The saturated acid was then dehydrogenated to linoleic acid by incubation with C. vulgaris, an organism which

I H-+ H

H-S H-7

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

2768 Biohydrogenation of Unsaturated Fatty Acids. V Vol. 246, No. 9

TABLE III

Ratios of 3H:‘YT of stearic acid and linoleic acid isolated after

incubation with Chlorella vulgaris Incubation conditions and isolation of the fatty acids are de-

scribed under “Experimental Procedure.”

Experiment

1 10.7 7.4 2 10.0 6.5

% 69 65

stereospecifically desaturates stearic acid to linoleic acid by removal of the n-hydrogens from carbons 9, 10, 12, and 13 (20). Thus, if the hydrogen was added at carbon 13 in the n-configura- tion by linoleic acid isomerase as shown in Fig. 1, the ratio of 3H :nC in the reisolated linoleic acid would be 75 y0 of the ratio of the stearic acid added to C. vulgaris. If, on the other hand, hydrogen added to carbon 13 in the L configuration (Fig. l), then the expected isotope ratio in the linoleate would be 25% of that in the stearate.

The results of two experiments (Table III) indicate that the hydrogen added to carbon 13 by linoleic acid isomerase is in the n configuration.

Mechanism of Isemerization-In previous studies with whole cells incubated in deuterium oxide, it was shown that the hy- drogen added at carbon 13 was derived from water (2). Similar studies, conducted with the purified isomerase, gave essentially the same results. Moreover, it was observed that the per- centage of deuterium incorporated at carbon 13 was the same (88%) after 18% as after 38% isomerization of the substrate had occurred. These results led to the proposal that the isom- erization involves the addition of a proton either directly from water or from some group in rapid equilibrium with water. It was concluded, therefore, that the isomerization of linoleic acid could result from either a successive hydration and dehydration of the fatty acid or from the addition and loss of a proton.

A hydration-dehydration mechanism would imply that 12-hy- droxy-cis-9-octadecenoic acid (ricinoleic acid) is an intermediate which is subsequently dehydrated to yield k-9, tr ans-ll- octadecadienoic acid, the observed product. However, no evi- dence of isomerization was obtained during incubation of a 24 MM or a 48 PM solution of either the naturally occurring n-ricin- oleic acid or a synthetically prepared racemic mixture with linoleic acid isomerase. The argument that inhibition of isom- erization by the D isomer might have prevented the detection of isomerization of the L isomer is negated by the fact that neither n-ricinoleic acid (4) nor the racemic mixture inhibited the isomerization of linoleic acid. In an effort to detect an enzyme-bound hydroxy acid intermediate, 1 mg of linoleic acid isomerase was incubated with 30 nmoles of 1-14C-linoleic acid (0.5 PCi) for 3 hours. With 0.2 mg of enzyme and 70 nmoles of linoleic acid, equilibrium (Keg = 60) (2) is reached in less than 10 min. No radioactivity coincident with a ricinoleic acid spot could be detected when half of the fatty acid incubated was sub- jected to thin layer chromatography.

Possible isotope effects in the breaking of the carbon-hydrogen bond at carbon 11 were investigated through the use of syn- thetically prepared 11, ll-dideuterolinoleic acid and perdeutero- linoleic acid isolated from algae grown in deuterium oxide.

TABLE IV Inhibition constants of competitive inhibitors of linoleic

acid isomerase

The incubation conditions were the same as those described in Table II except that the cuvette contained the inhibitor at the concentrations indicated. Calculations of Ki were derived from each apparent K, obtained by a computer program. Each value is derived from dunlicate determinations obtained from the aver- age of at least three replications.

Inhibitor

cis-9-Octadecenoic acid

cis-6-Octadecenoic acid

Linoleyl alcohol

Linoleyl amine Linoleyl amide

[II Ki

M x 106 M x 106

24 14.9 48 12.0 24 13.0 48 7.5 25 11.2 50 9.2 25 14.7 24 10.9

When either of these acids was used as the substrate, the rate of isomerization was about 50% of that obtained with nonlabeled linoleic acid. Kinetic analysis of the reaction with the deuter- ated substrates revealed that the presence of deuterium had not altered the Km but had reduced the VmaX by about 60% (Table II).

Two studies involving exchange of hydrogen at carbon 11 were conducted. In the first, attempts were made to detect incor- poration of deuterium into linoleic acid following incubation of this acid with the isomerase in deuterium oxide. No exchange in excess of that expected from the equilibrium of the isomeriza- tion reaction was observed. In the second study, no loss of deuterium from 24 PM 11 , 11-dideuterooleic acid was detected after incubation of this competitive inhibitor with the isomerase for 3.5 hours.

Kinetic Analysis of Inhibition of Linoleic Acid Isomeruse-We have reported (4) that cis-9-octadecenoic acid, cis-6-octadecenoic acid, linoleyl alcohol, linoleyl amine, and linoleyl amide all appear to be competitive inhibitors of the isomerization of linoleic acid. Further examination of these data indicates that the inhibition constants (KJ of these inhibitors are essentially equal in value to the K,,, (10 PM) for linoleic acid (Table IV).

DISCUSSION

In the previous paper describing studies of inhibition of linoleic acid isomerase by unsaturated fatty acids and analogues (4), three parameters were indicated to be involved in the bind- ing of substrate to the enzyme: (a) interaction of the r system of the double bonds, (b) hydrogen bonding of the substrate carboxyl group, and (c) hydrophobic interaction. The sub- strate specificity (i.e. requirements for isomerization) is even more stringent, requiring a cis-9,cis-12-diene system and a free carboxyl group. We have now shown marked substrate speci- ficity for the hydrocarbon end of the molecule; thus, an B-car- bon straight chain acid is the preferred substrate. These findings strengthen our conclusion that the physiological sub- strate for linoleic acid isomerase of Butyrivibrio $bl-isolvens is linoleic or linolenic acid.

Inhibition of linoleic acid isomerization by the trans isomers of linoleic acid led to the suggestion that the cis-9,&-12-diene

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Issue of %Iay 10, 1971 C. R. Kepler, W. P. Tucker, and X. B. Tove 2769

system induced a conformational change in the enzyme permit- ting isomerization (4). Similarly, the present finding that cis-9, cis-12-eicosadienoic acid, although inactive as a substrate, is inhibitory, suggests that the w chain also participates in inducing the active conformation of the enzyme.

The rapid incorporation of a proton from water at carbon 13 of the substrate led to consideration of the mechanism of the isomerization reaction as a hydration-dehydration sequence or as a direct isomerization involving a protic mechanism. Evi- dence against the hydration-dehydration mechanism is provided by the observations that neither enantiomer of ricinoleic acid was dehydrated to the conjugated acid and that radioactive ricinoleic acid was not detected when V4C-linoleic acid of high specific radioactivity was isomerized. The latter observation is signif- icant in view of the fact that an isotope effect was demonstrated with 11,l I-dideuterolinoleic acid, identifying the suggested dehydration reaction as rate-limiting. Thus, one might expect an accumulation of an enzyme-bound hydroxy acid intermediate. However, because the enzyme was not pure, the existence of an enzyme-bound 12.hydroxyoctadecenoate not in equilibrium with exogenous ricinoleate cannot be rigorously eliminated.

Three possibilities for a direct isomerization mechanism have been considered. These are diagrammed in Fig. 2 as (a) a carbonium ion intermediate, (b) a carbanion intermediate, and (c) a concerted reaction. A carbonium ion intermediate (Fig. 2A) would arise following stereospecific protonation of carbon 13 of the enzyme-bound substrate, with subsequent elimination of a proton from carbon 11 yielding the &s-9, tram-1 1-octadecadienoic acid product. According to this mechanism, the breaking of the carbon-hydrogen bond at carbon 11 would not be rate- limiting and, therefore, substitution of deuterium for hydrogen at carbon 11 should not affect the rate of isomerization. Since kinetic studies with the deuterated substrates showed no in- crease in K, but a decrease in the maximum velocity, the carbonium ion mechanism can thus be eliminated.

The carbanion mechanism (Fig. 2@ requires abstraction of a proton from the methylene bridge of linoleic acid, leaving an enzyme-bound carbanion to be protonated at carbon 13. Dis- sociation of the ion from the enzyme surface prior to neutraliza- tion can be disregarded since the addition of hydrogen at carbon 13 is stereospecific and because the products would be an equimolar mixture of cis-9, trans-1 I-octadecadienoic acid and trans.lO,cis-12-octadecadienoic acid as is the case with alkali- catalyzed isomerization (21). The latter isomer has never been detected following isomerization of linoleic acid by linoleic acid isomerase.

In an effort to demonstrate an enzyme-bound carbanion, we attempted to detect an enzyme-catalyzed exchange of deuterium for a carbon 11 hydrogen in linoleic acid and the loss of deuterium from 11 , 11-dideuterooleic acid. Since hydrogen exchange was not observed with either acid, evidence for an enzyme-bound carbanion was not obtained. Unfortunately, however, the absence of exchange does not necessarily eliminate a carbanion mechanism. Detectable exchange of deuterium with linoleic acid might not be expected since the collapse of the carbanion to the energetically favored cis, trans-conjugated isomer is likely to be faster than the combined rates of exchange of the enzyme- bound proton with a deuteron and reprotonation of the car- banion at carbon 11. Similarly, exchange of deuterium for hydrogen at carbon 11 of 11 ,ll-dideuterooleic acid might not be

FIG. 2. Protic mechanisms of isomerization by linoleic acid isomerase. A, carbonium ion intermediate; B, a carbanion inter- mediate; and C, a concerted reaction. Counting from right to left, carbon atoms 11, 12, and 13 are diagrammed.

expected since the carbon 11 hydrogens of linoleic acid are more acidic then the carbon 11 hydrogens of oleic acid.

Thus, it is clear that neither a carbonium ion mechanism nor a carbanion mechanism in which there is a dissociation of the ion from the enzyme surface is compatible with the data. We con- clude, therefore, that the isomerization involves either a con- certed reaction or an enzyme-bound carbanion in which pro- tonation occurs prior to dissociation from the enzyme surface. Conceptually, the only difference between the stereospecific protonation of an enzyme-bound carbanion and a concerted reaction involving the removal of a proton from carbon 11 and the addition at carbon 13 (Fig. 2C) is one of relative rate con- stants. A similar mechanism has been proposed by Rando and Bloch (22) and Helmkamp and Bloch (23) for the isomerization of /I, y-decenoate to a, fl-decenoate by P-hydroxydecanoyl thioester dehydrase. Endo, Helmkamp, and Bloch (24) have shown recently that P-hydroxydecanoyl thioester dehydrase isomerizes 3-decynoate to 2,3-decadienoate, which accounts for the marked inhibition of the enzyme by 3-decynoate (23). This enzyme differs from linoleic acid isomerase, however, in that fl-hydroxydecanoyl thioester dehydrase catalyzes an observable hydration and dehydration of a fatty acyl thioester as well as allene formation. In contrast, linoleic acid isomerase does not produce a detectable hydroxy acid and does not catalyze the formation of an allene from cis-9-octadecen-12.ynoic acid (crepenynic acid). Furthermore, it is only slightly inhibited by this acetylenic acid (4).

Kinetic analysis of the linoleic acid isomerase reaction has been facilitated by the fact that the reaction involves only a single substrate and product. Since 11 , 1 I -dideuterolinoleic acid, perdeuterolinoleic acid, and A6f9J2-octadecatrienoic acid have the same Michaelis constants but different maximum velocities (Table II), the kinetic analysis is simplified even further in that the reaction follows the classical theory of Michaelis and Menten. Thus, the Michaelis constant represents in effect the dissociation constant of the enzyme-substrate com- plex.

The electrophile that interacts with a double bond of a sub-

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Biohydrogenation of Unsaturated Fatty Acids. V Vol. 246, No. 9

FIG. 3. Molecular models of linoleic acid (left) and A~.s,~Z-

octadecatrienoic acid (right) in the extended and loop conforma- tions.

strate (or inhibitor) and the group that is hydrogen bonded to the carboxyl group of the substrate represent two relatively fixed points at the active center of the enzyme. It is difficult to imagine that the cis-6-octadecenoic acid and the cis-9-octa- decenoic acid lie fully extended in a hydrophobic pocket between these groups, particularly since the inhibition constants of these acids are essentially equal (Table IV). It seems more reasonable to picture the two binding groups at the active center lying in close proximity, forcing the fatty acid ligand to form a loop, the size of which depends on the distance between the double bond and the carboxyl group.

The presence of a loop in the substrate molecule is also com- patible with the Michaelis constants obtained for linoleic acid and A6~s~1z-octadecatrienoic (y-linolenic) acid. Molecular mod- els show that the extended conformation of linoleic acid and y-linolenic acid have considerably different shapes (Fig. 3) and, therefore, it would be predicted that these two substrates would exhibit different Michaelis constants. However, in a loop, the shape of the 2 molecules is very similar so that such a conformation appears to be more consistent with the identity of their Michaelis constants (Table II).

Since the Km for linoleic acid appears to reflect primarily the dissociation constant for the enzyme-substrate complex, its equality with the inhibition constants of the linoleate derivatives (Table IV) indicates that the alignment of each of these inhibitors at the active center of the enzyme must be very similar, if not identical, to that of the substrate. This is especially significant in the case of three inhibitors (linoleyl amine, linoleyl alcohol, and linoleyl amide) in that it implicates the involvement of the carboxyl group in the isomerization reaction. There are two reasonable explanations for such involvement of the carboxyl group: (a) the carboxyl group may induce a specific conforma- tional change in the enzyme, permitting isomerization to take place, or (b) the carboxyl group may be involved directly in the

FIG. 4. Proposed model for the isomerization of linoleic acid by linoleic acid isomerase. Pictured at the active site are an ele& tronhile (E) that interacts with one of the substrate double bonds. and two basic centers, one of which (B) is hydrogen-bonded to the carboxyl group of the substrate and the other (B-H) which serves as the donor for the hydrogen added at C-13.

reaction. Although there is no direct evidence which permits distinction between these two alternatives, we favor the direct involvement of the carboxyl group for the following reasons. Linoleate derivatives containing a carbonyl oxygen but lacking an active hydrogen, i.e. linoleyl aldehyde, linoleyl methyl ketone, and methyl linoleate, do not inhibit. Consequently, the hydrogen bond to the enzyme would appear to involve the hydrogen of the undissociated carboxyl group and a center of high electron density on the enzyme. Under these circumstances interaction between the substrate and enzyme through such a hydrogen bond is not expected to be appreciably different from that of the strongly inhibitory linoleate derivatives. Specific conformational changes resulting from a difference in size between the carboxyl group and the amide group appear unlikely since the size of these two groups is very similar. Moreover, if a specific conformational change were induced by the carboxyl group, it is unlikely that the enzyme would have the same affinity for linoleate as for linoleyl amide, linoleyl alcohol, and linoleyl amine (Table III), particularly in view of the lack of affinity of the enzyme for the aldehyde, methyl ketone, and methyl ester derivatives of linoleic acid. Finally, of all of the groups under consideration, only the carboxyl group is capable of serving as intermediary in a concerted exchange of protons in which the hydrogen bond to the enzyme would be broken, thereby permitting the release of the isomerized product.

Although tentative, a model of the mechanism as diagrammed in Fig. 4 is consistent with these considerations and with all of the findings to date. We envision the following sequence of events: initial binding of substrate to the enzyme in a hydro- phobic pocket which involves interaction of the a-electrons of a substrate double bond with an electrophilic group (-8’) on the enzyme and hydrogen bonding of the undissociated carboxyl group of the substrate with an electronegative center (B) on the enzyme. If the ligand is an B-carbon straight chain fatty acid containing a cis-9 ,cis-12-diene system, then induction of the proper conformation of the enzyme-substrate complex results in a concerted abstraction of a proton by the substrate carboxyl group from carbon 11 and a stereospecific attack (D configuration) by the proton of a conjugate acid (B-H) of the enzyme on carbon 13 of the substrate. The product, cis-9, trtsns-11-octadecadienoic acid, then dissociates from the enzyme. The two basic centers of the enzyme subsequently revert to their initial states.

AcknowZedgments-The LKB mass spectrometric analysis was performed by Dr. George Wailer and Dr. Earl Mitchell of Okla- homa State University. We would like to thank Jody Keyser for capable technical assistance and acknowledge the many stimulating discussions with Dr. H. R. Horton.

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Issue of Xay 10, 1971 C. R. Kepler, W. P. Tucker, and S. B. Tove 2771

REFERENCES

1. KEPLER, C. R., HIRONS, K. P., MCNEILL, J. J., AND TOVE, S. B., J. Biol. Chem., 241, 1350 (1966).

2. KEPLER, C. R., AND TOVI-G, S. B., J. Biol. Chem., 242, 5686 (1967).

3. WILDE, P. F., AND DAWSON, R. M. C., Biochem. J., 98, 469 (1966).

4. KEPLER, C. R., TUCKER, W. P., AND TOVE, S. B., J. Biol. Chem., 246, 3612 (1970).

5. TUCKER, W. P., TOVE, S. B., AND KEPLER, C. R., J. Label. Compounds, 6, in press (1971).

6. TUCKER, W. P., TOVE, S. B., AND KEPLER, C. It., J. Label. Compounds, in press (1971).

7. TAYLOR, W. R., AND STRONG, F. M., J. Amer. Chem. Sot., 72, 4263 (1950).

8. CRAM, D. J., AND ALLINGER, N. L., J. Amer. Chem. Sot., 73, 2518 (1956).

9. ELOVSON, J., Biochim. Biophys. Acta, 106, 291 (1965). 10. SCHROEPFER, G. J., JR., AND BLOCH, K., J. Biol. Chem., 240,

54 (1965). 11. KEPLER, C. R., AND TOVE, S. B., in J. M. LOWENSTEIN (Ed-

itor), Methods in enzymology, Vol. XIV, Academic Press, New York, 1969, p. 105.

12. 13.

14. 15. 16. 17.

18.

19.

20.

21.

22. 23.

24.

CLEUND, W. W., Advan. Enzymol., 29, 1 (1967). L~YNE, E., in S. P. COLO’I\.ICK AND N. 0. KAPLAN (Editors),

Methods in enzymology, Vol. III, Academic Press, New York, 1957, p. 448.

CHEN, R. F., J. Biol. Chem., 242, 173 (1967). DOLE, V. P., J. Clin. Invest., 36, 150 (1956). PRIVETT, 0. S., AND NICXELL, J?l C., Lipids, 1, 98 (1966). HARRIS, It. V., HARRIS, P., .~ND JAMES, A. T., Hiochim. Bio-

phys. Acta, 106, 465 (1965). BLIGH, E. G., AND DYER, W. J., Can. J. Biochem. and Physiol.,

37, 911 (1959). OKITA, G. T., K.4Ba~.4, J. J., RICHARDSON, F., AND LEROY,

G. V., Nucleonics, 16, 111 (1957). MORRIS, L. J., HARRIS, R. V., KELLY, W., AND J,\MEs, A. T.,

Biochem. J., 109, 673 (1968). NICHOLS, P. L., JR., HERB, S. F., AND RIEMENSCHNEIDER,

It. W., J. Amer. Chem. Sot., 73, 247 (1951). RANDO, R. R., .IND BLOCH, K., J. Biol. Chem., 243, 5627 (1968). HI~GLMKAMP, G. M., JR., BND BLOCH, K., J. Biol. Chem., 244.

6014 (1969). ENDO, K., HXLMKAMP, G. M., JR., AND BLOCH, K., J. Biol.

Chem., 246, 4293 (1970).

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Carol R. Kepler, W. P. Tucker and S. B. Tove11-trans-ISOMERASE FROM BUTYRIVIBRIO FIBRISOLVENS∆12-cis,

∆PROTON ADDITION AND MECHANISM OF ACTION OF LINOLEIC ACID Biohydrogenation of Unsaturated Fatty Acids: V. STEREOSPECIFICITY OF

1971, 246:2765-2771.J. Biol. Chem. 

  http://www.jbc.org/content/246/9/2765Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/246/9/2765.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from