THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 249, No. 9, Issue of May 10, PP. 2833-2642, 1974

Printed in U.S.A.

Enzymatic Interconversion of Oleic Acid, lo-Hydroxyoctadecanoic Acid, and trans-A1O-octadecenoic Acid

REACTION PATHWAY AND STEREOSPECIFICITY *

(Iteceived for publication, August 20, 1973)

a CHARLES E. MORTIMER~ AND WALTER G. NIEHAUS, JR.!

From the Department of Biochemistry, The Pennsylvania State Univekty, University Pa&,. Pennsylvania 16806

SUMMARY

A soluble enzyme preparation from Pseudomonas sp. NRRL B-3266 catalyzes the interconversion of cis-A9-octa- decenoic acid (oleic acid), trans.Alo-octadecenoic acid, and lo- n-hydroxyoctadecanoic acid. Oleic acid and lo-D-hydroxy- octadecanoic acid are directly interconvertible by hydration and dehydration. The cis and trans unsaturated fatty acid isomers are interconvertible by direct isomerization of the double bond. trans-A’O-Octadecenoic and lo-n-hydroxy- octadecanoic acid do not undergo direct interconversion without the intermediate formation of (enzyme-bound) oleic acid. Both hydration and isomerization reactions are very specific for a cis-A9 double bond, but the isomerization reac- tion is much more sensitive to changes in the hydrocarbon tail of the substrate distal to the double bond. The isom- erization proceeds stereospecifically, with the 1 I-L (Pro-R) hydrogen of oleic acid being removed and a hydrogen from the medium being added at carbon 9 in the L (Pro-R) con- figuration. In the reverse reaction the 9-L (Pro-R) hydrogen is removed and a hydrogen from the medium is added at carbon Il. Primary kinetic isotope effects are seen for the removal of the 9-L and 11-L hydrogens. There is a marked discrimination against tritium in the addition of the 11.~ hydrogen from the medium. The hydration of oleic acid and the isomerization of trons-A1o-octadecenoic acid to oleic acid proceed more slowly in highly enriched *HZ0 than in H20, but the isomerization of oleic acid to trans-A1o-octa- decenoic acid is slightly faster in 2Hz0.

An enzyme preparation from a l~seudomonatl has been shown

to catalyze the following reactions: hydration of oleic acid to

IO-D-hydrosyoctadecanoic acid (1, 2) ; hydration of palmitoleic

* This work was supported by (irants MI1 15788 and III> 01505 from the National Institutes of IIealth.

t: This report has been taken in part from the dissertation sub- mitted to the faculty of The Pennsylvania State University in partial fulfillment of the requirements for the Ph.!-). degree.

§ To whom correspondence should be addressed.

acid to IO-D-hydroxyhexadecanoic acid (2) ; hydration of linoleic

acid to 10-r-hydroxy-A12-cis-octadecenoic acid (3) ; and hydra-

tion of cis- and frans-9, IO-epoxyoctadccanoic acids to threo- and erythro-9, IO-dihydroxyoctadecanoic acids (4, 5). It was

also noted (2) that oleic acid was converted to trans-A1O-octa- deccnoic acid in low yield. We report here some further studies

of the mechanism of formation of this trans isomer of oleic acid.

We report the absolute stereochemistry of hydrogen addition

and abstraction at carbon 9 and carbon I1 during the conversion of oleic acid to Iruns-Alo-octadecenoic acid and during the reverse reaction. Preliminary accounts of this work have been pre-

sented (6-8).

MATERIALS AKD METHODS

Growth of Organism attd Preparations of Enzyme E&act-The organism used as a source of enzyme, Pseudo~~ot~as sp. NRRL 11-3266, was received from the Northern Regional Research Lab- oratory, Peoria, Ill. It was grown in a New Brunswick F-130 Fermentor on a medium consisting of 0.5yo potassium phosphate (pH 6.8), lyG glucose, 17; yeast extract, l”/b Bacto-tryptone, 0.1% MgSOd, and 0.030/, oleic acid. The cells were harvested by con- tinuous centrifugation and stored frozen until used. A soluble enzyme preparation was prepared by sonication of the cells in 0.02 M Tris-HCl buffer, pH 7.0, followed by centrifugation at 20,000 and at 110,ooO X 9 for 90 min each. The 110,000 X g super- natnnt had a protein concentration of 10 to 15 mg per ml. This was treated with 0.1 volume of a 2% solution of protamine sulfate, pH 7.0. The crude protamine sulfate supernatant was used as the enzyme source in most experiments reported herein. It was stored frozen and was stable for several months.

12adiochro7,~utographic Assa~-[1-14C]Oleic acid was purchased from New lingland Nuclear Corp. Unlabeled oleic acid was pur- chased from Xupelco, Inc., Bellefontc, Pa.

The assay for oleic acid isomerization involved incubation of (l-14C]oleic acid (0.04 to 0.1 mh%) in the appropriate buffer with varying amounts of enzyme extract. At appropriate times sam- ples were removed from the incubation mixture into hydrochloric acid to stop the reaction. The fatty acids were extracted with ether containing 10% methanol, dried over anhydrous MgSOc, and methylated with diazomethane. The products of the reac- tion were separated by low temperature argentation thin layer chromatography (Silica Gel G impregnated with 37, AgN03 using a solvent of pentane-ether (96:-1) at 8”). Chromatographic stand- ards of methyl oleate, methyl elaidate, and methyl 10.o-hydroxy- octadecanoate were used, and visualized by spraying with 0.2% 2’,7’-dichlorofluorescein in ethanol and viewing under ultra- violet light. The spots were marked and scraped into scintillation vials using a semiautomatic device obtained from Hansvedt Corp.,

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Urbana, 111. Scintillation counting was carried out in a Beckman LSZOOB.scintillation spectrometer ising a scintillation fluid con- taining. bv volume. 60% toluene. 30% Triton X-100 (Rohm and Haas), 10% water; aii 0.45yo ‘dipl&yloxazole. The radioac- tivities were used to determine the percentage of the total fatty acids present in each fraction, and initial velocities of formation of products were calculated.

For assays involving counting of products labeled with both 3H and 14C, adjustable energy channels of the Beckman LSSOOB were used, and efficiencies were determined using internal stand- ards of [3H]- and [‘4C]toluene.

&lass Spectral Analysis-Mass spectra were obtained using the LKB 9000 combination gas chromatograph-mass spectrometer (courtesy of the Department of Food Science, The Pennsylvania State University). The column used was 4yo SE-30 on Supelco- port.

Synthesis of Unsaturated Fatty Acid Substrates--The following unsaturated fatty acids were synthesized in both unlabeled and l-l% form: heptadec-Q-enoic; nonadec-Q-enoic; eicos-Q-enoic; heptadec-%enoic, cis- and trans-octadec-10.enoic. The procedure followed was essentially that of Ahmad and Strong (9) involving coupling of an w-chloroalkyliodide with a terminal acetylene, formation of the nitrile bv condensation with KCN or K’%N. hydrolysis of the nitrile ti yield the acetylenic fatty acid, and hydrogenation to the cis-olefinic fatty acid using a Lindlar cat- alyst. All fatty acids used in these experiments were chromato- graphically and radiochromatographically pure (>95%) based on silica gel thin layer chromatography, argentation thin layer chro- matography, and gas-liquid chromatography on 40/, SE-30 and 10% ethylene glycol succinate using a Barber Colman 5000 instru- ment equipped with a stream splitter and radioactivity monitor.

Reagents for the syntheses were obtained from Aldrich or K & K. Lipids and chromatographic materials were obtained from Supelco.

Stereospecijic Synthesis of Trans-A’O-[9-n-3H]- and trans-A1O- 19-L-3H10ctadecezoic Acids-In order to determine the stereo- specificity of the enzymatic interconversion of cis-A9- and frans- A’O-octadecenoic acids, we needed samples of substrates labeled singly and stereospecifically with tritium. These may be prepared from the corresponding optically active hydroxy fatty &ids by reduction of the corresponding tosylates with LiA13Hd as was described bv Schroenfer and Bloch (10). The resultant tritium-labeled oct,adecanoiE acid samples were’converted to the desired labeled unsaturated fatty acids by microbiological desat- uration.

The synthetic procedure followed was essentially that used by Schroepfer and Bloch (10). Optically pure Q-n-hydroxy-A’2- octadecenoic acid was isolated from seeds of Dimorphothica sinuata which were graciously provided by Dr. R. G. Powell of the North- ern Regional Research Laboratory. A sample of seeds from Holarrhena antidysenterica was also obtained from Dr. Powell but the hydroxy fatty acid from these seeds was only 7597, of the D isomer and 25yo of the L isomer.

Fifteen grams of D. sinuata seeds were ground in a mortar and pestle and extracted in a Soxhlet apparatus with pentane. The solvent was evaporated and the oil was saponified by refluxing with methanolic KOH. The nonsaponifiable material was ex- tracted and discarded and the fatty acids were extracted after acidification of the reaction mixture. The fatty acids were con- verted to their methyl esters with diazomethane and the methyl 9-n-hydroxy-A12-octadecenoate was purified on an activated silicic acid column (Supelcosil ATF), eluted with pentane-ether (70: 30). The unsaturated hydroxy fatty acid methyl esters were hydro- genated over palladium black catalyst in methanol at 40 p.s.i.g. of hydrogen for 5 hours in a Parr hydrogenation apparatus. The resulting methyl Q-n-hydroxyoctadecanoate was purified by crystallization from acetone. Over-all yield was 3.5 g. The nonracemic nature of the product was determined by measure- ment of the optical rotation (see below).

To prepare Q-L-hydroxyoctadecanoate, the tosyl ester of methyl Q-n-hydroxyoctadecanoate was prepared and subjected to alka- line hydrolysis. Methyl Q-n-hydroxgoctadecanoate (1.3 g) was dissol;ed in 50 ml of freshly distilled pyridine and treated with 7.4 g of recrvstallized D-toluenesulfonvl chloride. The mixture wasstored ai 4” for 48 Ihours. Compl&eness of reaction was as- certained by thin layer chromatography on Silica Gel G with pentane-ether-acetic acid (50:50:1). The pyridine solution was

added to 2 volumes of water and extracted with ether. The tosylate was purified on a Supelcosil ATF column, being eluted with 10% ether in pentane. The yield was 1.44 g of methyl Q-D- tosyloctadecanoate.

A portion of the tosylate (1.0 g) was hydrolyzed with inversion by refluxing for 10 hours in 60 ml of aqueous 4 N NaOH plus 12 ml of dioxane. After extraction of nonsaponifiable material, the reaction mixture was acidified and the Q-L-hydroxy- octadecanoate was extracted with ether, methylated with diazo- methane, and purified by column chromatography in a yield of 0.25 g.

The optical rotations of both the methyl Q-n-hydroxy- octadecanoate and methyl Q-L-hydroxyoctadecanoate were meas- ured in spectral grade methanol using a Perkin-Elmer model P-22 spectropolarimeter (kindly provided by Dr. L. Jackman, Penn State) (Table I). The samples were concluded to be optically pure, based on comparison with literature values and with stand- ard lo-~- and lo-L-hydroxyoctadecanoate prepared from Pseu- domo,zas NRRL B3266. The tosyl ester of methyl Q-L-hydroxy- octadecanoate was prepared as described above.

A portion of each of the tosylates was reduced with LiA13H4 to form the corresponding tritiated octadecanols. Methyl Q-D- tosyloctadecanoate (4.7 mg, 10 pmoles) was dissolved in 1.5 ml of tetrahydrofuran, freshly distilled from LiAlHa. Working in a nitrogen-filled glove bag, 12.5 mCi of LiA13Hd (25 &Ji per patom of H) were added and tetrahydrofuran was added to a final volume of approximately 8 ml in the 15.ml reaction flask. The mixture was gently refluxed for 10 hours. To assure complete utiliza.tion of the LiA13Hr, an additional 10 mg of tosylate were added and refluxing was continued for 2 hours. Unlabeled LiAlH, then was added in excess and refluxing was continued another 2 hours. Excess LiAlH, was decomposed with ethyl acetate and the reac- tion mixture was acidified and extracted with ether. The tritiated octadecanol was purified by column chromatography on Supelcosil ATF, being eluted by 12% ether in pentane. Methyl Q-L-tosyl- octadecanoa.te was carried through an identical procedure.

The octadecanols were then oxidized to octadecanoic acid by chromic acid (11). The octadecanol (6 mg) was dissolved in 4 ml of glacial acetic acid to which were added 15 mg of chromic acid. The samples were allowed to react at room temperature for 5 hours. Three milliliters of water were added, followed by sufficient solid NaHS03 to reduce the excess chromic acid. The mixtures were extracted with ether and tritiated octadecanoic acids were purified by column chromatography. Since the LiA13Hd reduction is reported to result in reductive removal of the tosyl group with inversion of configuration by an &-a-type

TABLE I

Gas chromalographic retenliorl times of unsaturated fatty acid methyl esters ar,d their ozonolysis products; 10% ethylene glycol succinate

on acid-washed chromosorb W, 200”

Compound

Methyl oleate. Methyl cis-octadecenoateb.. . Methyl trans-octadecenoateb. Methyl a&elate semialdehyde.. Methyl adipate semialdehyde. Methyl undecandioate semialdehyde Methyl ester semialdehyde from cis-

octadecenoateb.. Methyl ester semialdehyde from trans-

octadecenoateb..

T,

Flame detector

Radioactivitya detector

986 986 983 653 277

1130

1035 1035 1032 702

650 699

852 900

a The lag of the radioactive peak relative to the mass peak is due to the length of tubing between splitter and detector and is constant at 48 to 50 s.

b Derived from incubation of [l*C]oleic acid with the Pseudom- onas enzyme.

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reaction (lo),, the products were expected to be [9-L-3H]octadec- anoic acid from the 9+tosyloctadecanoate, and [9-n-3H]octa- decanoic acid from the 9-L-tosyloctadecanoate. As will be mentioned under “Discussion,” the LiAlHa reduction does not proceed with total stereospecificity.

The tritiated octadecanoic acids were converted to [9-3H]A10- octadecenoic acids by microbiological desaturation. A strain of Bacillus cereus was reported by Dart and Kaneda (12) to desat- urate stearic acid exclusively to cis-Alo-octadecenoic acid. How- ever, we were unable to obtain a culture of this organism from Dr. Kaneda. We obtained another strain of Bacillus cereus (PSU-43) from Dr. It. Doty (Department of Microbiology, Penn State) which produced both A5- and Ai0-octadecenoic acids and, by adjusting the culture conditions, we were able to produce nearly pure cis-Ai0-octadecenoate from added octadecanoate. The B. cereus was grown in 125-ml flasks containing 25 ml of 0.8q7c Difco nutrient broth plus 4yo glucose and O.OS’% ferric ammonium citrate. The culture was grown at 30” on a rotary shaker to an optical density (660 nm) of 0.5. Tritiated octadecanoic acid (400 pg) then was added in 0.5 ml of ethanol along with 0.25 ml of 0.5% ferric ammonium citrate. Incubation was continued for 3 hours. The cells were collected by centrifugation and digested with methanolic KOH. The fatty acids were extracted with pentane after acidification, methylated, and purified by preparative thin layer chromatography. The product was shown to be radio- chemically pure cis-octadecenoic acid by thin layer and gas chro- matography. Using [1-‘%]octadecanoic acid as a substrate for microbiological desaturation in a parallel experiment, the double bond was shown to be exclusively Ai0 by reductive ozonolysis (13).

The final step in the synthesis of the 9-n and 9-L-lrans-Aio- octadecenoic acids was chemical isomerization of the double bond (14). The methyl cis-A10-[9-o-aH]- and cis-A10-[9-L-3H]octa- decenoates were dissolved in 5 ml of glacial acetic acid to which was added dropwise with stirring 1 ml of 20% NaN02. After 30 min at room temperature, 5 ml of water were added and the fatty acid methyl esters were extracted with ether. The trans isomers were purified by prepara.tive argentation thin layer chromatog- raphy and saponified with methanolic KOH to yield trans.Ai0- [9-rGH]octadecenoic acid and trans-A10-[9-L-3H]octadecenoic acid.

Stereospecijk Synthesis of [ll-L-3H]Oleic Acid-A sample of 11-n-hydroxyoctadecanoic acid (a generous gift of Dr. M. Ham- berg, Karolinska Institute) was converted to [ll-L-~H]- octadecanoic acid by procedures essentially identical-to those described above. The microbiological desaturation to oleic acid was accomplished using a culture of Corunebacterium diphtheriae Crs(-) to;(-) (a gift of Dr. A. M. Pappenheimer, Harvard Uni- versitv). as described bv Schroenfer and Bloch (10). The final

“ , ,

product was characterized by radio thin layer and ‘gas chroma- tography and the double bond position was confirmed using [l-l%]- octadecanoic acid and reductive ozonolysis.

RESULTS

unsaturated fatty acid as cis-A9-octadecenoic acid. The ozonol- ysis product from the truns fraction was identified as the lo- carbon semialdehyde by comparison of its retention time with the 5-, 9-, and 11-carbon standards. Thus the truns unsaturated fatty acid produced by the Pseudomonas enzyme is trans-AlO- octadecenoic acid.

The double-bond positions of the cis and trans fractions were also determined by the mass spectral method of Niehaus and Ryhage (15). The unsaturated fatty acids were oxidized by alkaline permanganate to dihydroxy fatty acids which were characterized by thin layer radiochromatography on boric acid- impregnated plates (16). The dihydroxy fatty acid formed from the cis fraction was exclusively the ergthro isomer, that formed from the trans fraction was exclusively the threo isomer. Gas chromatographic-mass spectral analysis gave characteristic fragments at m/e 201 and 157 for derivatives of standard oleic acid and the cis fraction. The corresponding values were 215 and 143 for the trans isomer (Table II). These values show that the double bond of the truns-octadecenoic acid is located between Cl0 and Cii. Other peaks at m/e 151 and 183 are also characteristic of this double bond location (15). Thus the enzymatic product has been unequivocally identified as trans- AiO-octadecenoic acid.

Mechanism of Production of trans-A1o-Octadecenoicc Acid-The production of trans-Ai0-octadecenoic acid from oleic acid could occur via two potential pathways (Scheme 1) : direct isomeriza-

TABLE II Mass spectral identification of methyl lO,li-dimethoxyoctadecanoate

m/e I Relative intensity

29 I 27 41 43 45 55 58 59 67 69 71 81

143 151

I 21

157 5

I 52 41 94 63 35 27 23 92 79 25 39

IdentiJication oj trans Isomer as trans-A’O-Octadecenoic Acid- To prepare sufficient trans isomer for identification, 1 mg (3.5 pmoles) of oleic acid was dissolved in 0.1 ml of ethanol and 0.01 N NaOH was added to neutralize the carboxylate group. This mixture was incubated in 20 IrIM acetate buffer, pH 6.0, with 60 mg of enzyme protein at room temperature for 15 min.

The fatty acids were extracted and separated by preparative argentation thin layer chromatography. Double-bond positions of the cis and trans fractions were determined by reductive ozonolysis (13) and the fragments were identified by gas-liquid chromatography on 10% ethylene glycol succinate on acid washed Chromosorb W (Supelco) employing both mass and radioactivity detectors. The ozonolysis was performed using the Supelco microozonizer. Standards of methyl oleate, methyl elaidate, methyl vaccenate, and methyl petroselenate were used t.o identify the methyl ester semialdehydes. The results (Table I) show that both the cis and trans unsaturated fractions con- tained only octadecenoic acid. The ozonolysis product from the cis fraction was the g-carbon semialdehyde, thus identifying this

183 24 201 9 215 100

,F=“c \H

HOOC-(CH,), ;-KH,),-CH,

Isomerizotion

Hydration

(

\

‘-60 HOOC-(CH,),-: H’c$

HP /L H \

Y (CH,), CH,

/ Dehydration

H yHH

/

HOOC-(CH,),-;-$-;-(CH,),-CH,

SCHEME 1

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tion of the &s-A9 double bond; or adclition of water across the c&-A9 double bond, and subsequent removal of the elements of water from carbons 10 and 11. To distinguish between these possibilities the following experiments were performed.

[lJ4C]Oleic acid (0.45 pmole, 1.1 pCi) and [lo-DL-3H]-[10-~~-

h@oz2/]octadecanoic acid (0.45 pmole, 2.4 PCi) (prepared by reduction of lo-ketooctadecanoic acid with NaB3H4) were in- cubated with 20 mg of enzyme protein in 25 ml of potassium phosphate buffer, 0.1 M, pH 5.6. Samples were removed at intervals from 10 s to 2 hours and the products were extracted and separated. The change in fatty acid composition and 3H:14C ratio with time is shown in Fig. 1. The 3H:14C ratios of oleic acid, truns-Aro-octadecenoic acid, and lo-hydroxyoc- tadecanoic acid at 1 and 2 hours (not shown) were: 0.9, 1.0, and 1.3; and 1.1, 1.1, and 1.1, respectively. Since the reaction had reached isotopic equilibrium, it had presumably also reached thermodynamic equilibrium. Under these conditions of in- cubation, therefore, equilibrium was reached at 67% hydroxy- octadecanoic acid, 8% oleic acid, and 25% frans-ArO-octadecenoic acid. The fact that the 3H:14C ratio of [runs-ArO-octadecenoic acid closely parallels that of oleic acid throughout t.he course of the incubation strongly implicates oleic acid as its immediate precursor. A minor contribution of the hydration-dehydration pathway, possibly involving an enzyme-bound intermediate, cannot be rigorously eliminated, however.

An alternative method to investigate this question therefore was employed whereby trans.AlO-octadecenoic acid was incu- bated with enzyme in 2Hz0 enriched medium, and the resulting IO-hydroxyoctadecanoic acid was analyzed for its deuterium content by gas chromatography-mass spectrometry. Exam- ination of the two potential pathways (Scheme 1) shows that a direct hydration of the truns-A1O double bond would result in the stable incorporation of one atom of ,deuterium at carbon 11,

2

y, 00 2 4 ‘6 * 10 0 2 4 6 0 10

MINUTES MINUTES

FIG. 1. A, time course of interconversion of [14C]oleic acid (o), [3H]10-hydroxyoctadecanoic acid (A), and trans-A1O-octadecenoic acid (0). Oleic acid (0.018 mnl) and hydroxyoctadecanoic acid (0.018 mM) were incubated with enzyme (1 mg of protein per ml) in 0.1 M phosphate buffer, pH 5.6. Correction has been made fol the unreactive lo-L-hydroxyoctadecanoic acid present in the incubation. The quantity of each product per ml of incubation mixture was assayed radiochromatographically. B, 3H:14C ratio of the products of the incubation of [r4C]oleic acid and [3H]hy- droxyoctadecanoic acid. Oleic acid (0) ; Irarls-A1o-octadecenoic acid (0); hydroxyoctadecanoic acid (A). Correction has been made for the unreactive IO-n-[3H]hydroxyoctadecanoic acid pres- ent in the incubation. A logarithmic scale has been used for presentation of the first 10 min of the 2-hour incubation. Values

whereas the isomerization-hydration pathway would result in the stable incorporation of 2 atoms of deuterium, one at carbon 11 during isomerization, and a second at carbon 9 during hy- dration. Therefore, analysis of the distribution of deuterium in the hydroxyoctadecanoate, especially after very short incu- bation times, should allow one to assess the extent to which direct hydration of the trans-Alo double bond might occur. Several technical difficulties arose during this experiment. It was found (Fig. 2) that the conversion of truns-Ar0-octadecenoic acid to IO-hydroxyoctadecanoic acid occurs much more slowly in 2Hz0 than in lHzO, necessitating the use of relatively large amounts of enzyme protein. This, coupled with the need to analyze very early time points at which extremely small quan- tities of lo-hydroxyoctadecanoate had been produced, cause us to be concerned with possible contribution from endogenous fatty acid present in the enzyme preparation, The crude en- zyme preparation, containing 25 pg of fatty acid per mg of pro- tein, was added to 15 volumes of acetone at -15”. The re- sulting protein powder contained only 0.25 I.rg of fatty acid per mg which did not interfere in the subsequent analyses.

The acetone-precipitated enzyme (6 mg) was dissolved in a medium containing 0.1 ml of 0.1 M phosphate buffer (pH 6.1) and 19.9 ml of 2Hz0 (99.8 atoms y0 excess) (Bio-Rad), yielding a calculated over-all enrichment of 99.2oj,. The mixture was allowed to equilibrate at 10” for 30 min. A sample was removed to assay for contributions from cndogenous fatty acids. A sample of trans-A10-[l-14C]octadecenoic acid (4.3 pmoles in 10 ~1 of pentane) was added to the enzyme buffer mixture and incubated at room temperature with stirring. Samples were removed at 4, 10, 25, and 75 min and the fatty acids were ex- tracted and separated by preparative thin layer chromatog- raphy. The product distribution was determined by radio- chemical assay. The methyl ester of lo-hydroxyoctadecanoic acid was converted to its trimethylsilyloxy derivative by treat- ment with 10 ~1 of N,O-bis(trimethylsilyl)acetamide in 50 ~1 of pyridine 5 min prior to injecting the sample into the LKB 9000 gas chromatograph-mass spectrometer. Spectra were taken of derivatives of standard 10.hydroxyoctadecanoate and of the lo-hydroxyoctadecanoate samples from the 2Hz0 incubation. Spectra were also taken of derivatives of lo-hydroxy- octadecanoate derived from oleic acid in 2Hz0, which has been shown (17) to contain 1 atom of deuterium at carbon 9 in the L configuration. The major fragmentation pattern is shown in Scheme 2. The major peak at m/e 215 contains carbons 10 through 18 and therefore a deuterium atom at carbon 11 will increase this fragment to m/e 216. Likewise, the peak at m/e

273 represents carbons 1 to 10 and incorporation of a deuterium atom at carbon 9 will increase this fragment to m/e 274. At all time points examined, 97 y0 of the IO-hydroxyoctadecanoate molecules contain deuterium in t,he fragment Cr-Cro, presumably at CQ, and 96% of the molecules contain deuterium in the frag- ment Cl&&, presumably at Cl1 (Table III). Since even at

m/e 215

r- KHJ, Si

H6H H,COOC-(CH,),-C-C-C-(CH,),-CH,

H HIH . I

m/e 273 at later time points are found in the text. &IEME 2

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Mass spectral analysis of methyl-lo-trimethylsilyloxyocladecanoale derived from incubation of trans-Alo-octadecenoic acid in 2HzO

TABLE III TABLE IV

Substrate speciJcity of oleale hydratase-isomerase

Relative intensity Incubation

time octadecanoate m/e 215 1 m/e 216 1 m/e213 1 m/e 274

min 4 3.2 4.5 100 2.2 70

10 5.7 4.3 100 2.2 70 25 8.0 4.3 100 2.2 71 75 9.8 4.4 100 2.2 70

the earliest time points assayed (3% conversion to hydroxy- octadecanoate) no monodeuterated product is seen, we can conclude that direct hydration of the trans-Alo double bond does not occur to a measurable extent, and the reaction with which we are dealing is a direct isomerization of the &s-A9 and trans-Alo double bonds.

Substrate Specijicity for Hydration and Isomerization-It had previously been demonstrated that the Pseudomonas enzyme preparation would catalyze the hydration of oleic acid, pal- mitoleic acid (1, 2), and linoleic acid (3). No attempts were made to measure the relative rates of hydration of these various substrates, nor was the possibility of double-bond isomerization investigated for these other substrates. In an attempt to gain further insight into the structural features of the substrate re- quired for each of these enzymatic processes, we synthesized a series of cis monounsaturated fatty acids, labeled with 14C in the carboxyl group and determined the relative rates of hy- dration and isomerization. Since the rate of hydration is much greater than is the rate of isomerization, somewhat different

Substrate I

Hydration I

Isomerization

lG:lAs..... 17:1As...... 18:lA9.... 19:lAs...... 20:1~9.... 18:2 As,=. . 17:1As...... 18: 1 A*O-cis 18: 1 Ag-lrans

nmoles/min/ntg p*otein 250 85 85

100 17

150 17

n*toles/min/mg pro1ein

0.25 0.3 1.3 0.5 0 0 0 0 0

TABLE V

Mass spectral iclentijcation of methyl 9-trimethylsilyloxyhepta- decanoate derived jrom hydration of cis-As-hepladecenoic acid

m/e I Relative intensity

73 87 75 56 79 17 83 18

103 15 109 15 129 12 155 20 215 95 216 18 259 100 2G0 20

incubation conditions were used for the two assays. Both as- says were performed at pH 5.2 and room temperature, but for hydration or isomerization reaction when added to the incu- the isomerase assay somewhat lower substrate concentration bation mixture at the same concentration as the oleic acid sub-

strate. Interestingly, cis-A*-heptadecenoic acid was a sub- strate for hydration but not for isomerization. The hydration product has been identified as a 9-hydroxyheptadecanoic acid by gas chromatographic-mass spectral analysis of the tri- methylsilyloxy derivative (Table V). The major peaks at m/e

215 and 259 correspond to the peaks at m/e 215 and 273 in the spectrum of IO-trimethylsilyloxyoctadecanoate, as shown in Scheme 2. The absolute,stereochemistry has not been deter- mined, but is presumed to be D.

(0.04 versus 0.10 mM) and higher enzyme concentration (1.2 versus 0.12 mg per ml were employed). For the hydratase as- say, time points were taken at 0.5, 1, 2, and 4 min; for the iso- merase assay, time points were taken at 1, 2, 4, and 6 min. All assays were linear throughout this incubation period with the exception of the hydration of palmitoleic acid, which was sub- sequently assayed at 0.25, 0.5, 1.0, and 1.5 min.

The results (Table IV) show several interesting features of the reactions. Under the conditions of the assay, the rate of hydration of oleic acid (18: 1 Ag) is about 65 times the rate of its isomerization to the trans-Alo isomer. The effect of introducing changes into the hydrocarbon tail of the substrate, distal to the &s-A9 double bond, is quite different on the hydration and iso- merization reactions. For example, palmitoleic acid (16:l Ag) and linoleic acid (18:2 AsJz) are hydrated at 2 to 3 times the rate seen for oleic acid, but the rate of isomerization is greatly reduced for palmitoleic acid and is zero for linoleic acid. The isomerization of linoleic acid would presumably have produced a conjugated fatty acid, trans-Ar0, cis-A’*-octadecadienoic acid. A mixture of this compound and the cis-A9, trans-A11 isomer, produced by alkaline treatment of linoleic acid (18) was used

Stereospecificity of Hydrogen Abstraction during Isomerization-

To determine the stereochemistry of the hydrogen removed from the unsaturated fatty acid during isomerization, we in- cubated stereospecifically tritium-labeled fatty acids mixed with 14C-labeled fatty acid with the Pseudomonas enzyme prep- aration. From the 3H:14C ratio of substrate and product we could determine whether the labeled hydrogen had been re- moved during the enzymatic isomerization.

To approximately 40,000 dpm each of the trans-&0-[%w311]- and the trans-A10-[9-QH]-octadecenoic acid were added chem- ically synthesized trans-AlO-[1-14C]octadecenoic acid and a portion was removed to determine the 3H:14C ratio. The remainder of

as the chromatographic standard for the assay. The results each doubly labeled substrate was incubated with the Pseu- seen with the 17 : 1, 19 : 1, and 20 : 1 isomers are less striking but domonas enzyme preparation (1 ml of 0.1 M sodium acetate, pH also show greater relative rates of hydration than isomerization. 5.2, and 1.2 mg of protein) for 30 min. The incubations were Several other potential substrates, including elaidic acid (trans- terminated and the hydroxy, cis, and trans unsaturated fatty A9-octadecenoic acid), cis-AlO-octadecenoic acid, and 9- and acids were separated. The fatty acid methyl esters were elutcd lo-octadecynoic acids, underwent neither hydration nor isom- from the silver nitrate impregnated silica gel with 20% ether erization. The acetylenic analogs did not inhibit either the in pentane and were counted in the Beckman LS 200B liquid

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scintillation spectrometer using a scintillation fluid of 0.4% diphenyloxazole in toluene. Efficiencies were determined using internal standards of [3H]- and [14C]toluene.

Three possibilities exist for removal of a hydrogen from CS of truns-A10-octadecenoic acid: the D (Pro-S) hydrogen may be removed; the L (Pro-R) hydrogen may be removed; the reaction may proceed without stereospecificity, both D and L hydrogens being removed to some extent. For the first possibility, removal of the n-hydrogen, the cis-Ag-octadecenoate product derived from trun.s-A10-[9-n-3H]-octadecenoate would have a 3H :14C ratio of 0, and the cis-Ag-octadecenoate product derived from tran~-A~~- [9-GH]-octadecenoate would have the same 3H :14C ratio as the substrate. For the second possibility, L-hydrogen abstraction, the cis-Ag-octadecenoate derived from the 9-n-3H-labeled sub- strate would have the same 3H :14C ratio as the substrate, whereas the product derived from the 9-L-3H-labeled substrate would have a 3H:14C ratio of 0. For the third possibility, nonstereo- specific hydrogen removal, the product derived from both the n-Wlabeled and L-Wlabeled substrates would have a 3H:14C ratio significantly lower than that of the substrate. The results (Table VI, Experiments A and 13) are consistent only with the stereospecific removal of the 9-L (Pro-R) hydrogen during the isomerization of transd’o-octadecenoic acid to cis-Ag-oct.a-

decenoic acid. The finding that the 3H:14C ratio of the cis-A9 product derived from trans-A10-[9-L-3H]octadecenoic acid de- creased to 20% that of the substrate, and not to 0, is presumably due to a partially racemic nature of the stereospecifically. labeled substrate, and is not without precedent (19) (see “Discussion”). That the remaining 20% of the tritium is not due to a non- stereospecific abstraction of hydrogen is shown by the fact that there is no significant difference in the 3H:14C ratios of substrate and products when Iruns-A10-[9-n-3H]octadecenoic acid is the substrate. The increased 3H:14C ratio seen in the unreacted truns-A10-[9-L-3H]octadecenoic acid to 130% that of the initial

substrate ratio is indicative of a primary isotope effect in the

removal of the 9-L hydrogen.

conversion of cis-Ag-octadecenoic acid to truns-AlO-octadecenoic acid was investigated as described above, using cis-Ag-[ll-~-3H]- octadecenoic acid as substrate (Table VI, Experiment C). These data show that it is the 11-L (Pro-R) hydrogen which is abstracted during the isomerization of cis-Ag-octadecenoic acid to truns-

AlO-octadecenoic acid. The 3H:14C ratio of the product is again 20yo that of the substrate, indicative of a partially racemic nature of the substrate, and there is again a primary isotope effect for removal of the 11-L hydrogen.

The stereospecificity of hydrogen removal from Cl1 in the

Stereospecijicity of Hydrogen Addition at Carbon 9-Incuba- tions carried out in 2H20 enriched medium demonstrated that a hydrogen derived from the medium was incorporated during the isomerization reaction (Table III). In order to examine the hydrogen addition in more detail, we incubated cisdg-octa- decenoic acid in medium enriched with 3Hz0 and examined the extent of tritium incorporation and the stereochemistry of the addition. [1-14C]Oleic acid (1.6 kmoles, 0.098 &i per pmole) was incubated with 1.2 mg of enzyme protein in 0.2 ml of 3H20 enriched medium (2 pCi per patorn of hydrogen) for 9 hours. The long incubation time was necessitated by the use of the very high substrate concentration (8 mM). These conditions were chosen to obtain incorporation of significant amounts of tritium without requiring an excessively large total quantity of 3Hz0. The reaction was stopped and the products were ex- tracted and separated by preparative argent,ation thin layer chromatography. truns-A1O-Octadecenoate comprised 8.8 % of the total products or about 40 pg. Based on the known spe- cific activity of the [lJ4C]oleic acid, (0.098 PCi per pmole) and the tritium enrichment of the medium (2 $Zi per patom of hy- drogen), it could be calculated that the 3H :14C ratio of the truns-

Alo-octadecenoate should be 20.6. The experimentally deter- mined value was 19.4 (Table VII, A), indicating that isotopic equilibrium between tritium and protium was achieved over the long incubation period. The IO-hydroxyoctadecanoic acid produced from oleic acid during the incubation (84yo of the products) was also isolated and the 3H:14C ratio was determined (Table VII, A). The 3H:14C ratio was 85% of that calculated for the addition of a single hydrogen. Thus, very little dis-

TABLE VI

Stereospecificity of hydrogen removal during isomerization TABLE VII

A. Substrate trans-A10-[l-14C 9.w3H]Octa- decenoic acid. ’

Products cis-A9-Octadecenoic acid. . IO-Hydroxyoctadecanoic acid trans.Alo-Octadecenoic acid.

B. Substrate trans-A10-[l-*4C 9-L2H]Octa- decenoic acid. y

Products cis-A9-Octadecenoic acid. IO-Hydroxyoctadecanoic acid. trans-AlO-Octadecenoic acid

C. Substrate cis-A10-[l-14C, 11-GH]Octadec- enoic acid.

Products cis-A9-Octadecenoic acid. . trans-A’O-Octadecenoic acid.

100

11 18 71

0.578

0.553 96 0.558 97 0.650 112

100

12 19 69

0.884

0.183 0.180 1.140

100 4.80

28 8.34 0.97

vl:“C

21 20

129

174 20

Incorporation of tritium into fatty acids by incubation in 3HzO-enriched medium

Substrate W20

A. cis-A9-[l-14C]Oc- tadecenoic acid (0.098 pCi/ pmole)

B. cis-A9-[l-14C]Oc- tadecenoic acid (1.07 &i/Mmole)

C. trans-A10-[l-14C]- Octadecenoic acid (0.068 pCi/ rmole)

2.6

1.43

o Observed at 540 min. b Observed at 30 min. c Observed at 60 min.

Products

tru?~s-A10-18: 1 lo-Hydroxy-18: 0

trans-Alo-18: 1 lo-Hydroxy-18:O

cis-A9-18: 1 IO-Hydroxy-18:O

20.6 20.6

2.4 2.4

21.0 42.0

Observed

19.4s 17.4a

1.3*, 1.4c 1.26, 1.3~

1.P 16.7c

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crimination against tritium esists during the hydration of the &-A9 double bond. Schroepfer (9) has shown that this hydrogen is added at c&bon 9 in the L configuration.

The determination of the stereochemistry of the tritium added during isomerization is based on the finding by Schroepfer and Bloch (4) that in the conversion of stearic acid to oleic acid by Corynebacterium diphtheriae, the 9-n (Pro-S) and 10-n (Pro-S) hydrogens are stereospecifically removed. The frans-A10-[9-31-I]- octadecenoate derived from the aH20 incubation was diluted with unlabeled methyl stearate and methyl [ 1 -W]stearate and subjected to catalytic hydrogenation (Palladium black cata- lyst,, 40 p.s.i.g. of H?). The doubly labeled methyl stearate was saponified with methanolic KOH at room temperature overnight. The recovered stearic acid had a 3H:14C ratio of 2.14. The [l-14C,9-aH]stearic acid was added in 0.5 ml of ebhanol of a growing culture of C. diphtheriae as described above. The fatty acids were extracted and separated as described. The saturated and cis unsaturated fatty acid fractions were isolated and the 3H:14C ratio of each was determined (Table VIII). Since there is essentially no decrease (-6c/J in the 3H :14C ratio upon removal of the 9-n (Pro-S) and 10-11 (Pro-S) hydrogens, it is apparent that the tritium which was incorporated upon

conversion of cis-A9-octadecenoic acid to (runs-AlO-octadecenoic acid was of the 9-L (Pro-R) stereochemistry.

Since the need to produce a rather large quantity of the ~rans- AlO-octadecenoic acid had dictated an estremely long incubation time in the above experiment, we repeated the experiment under slightly different conditions to investigate further the extent of incorporation of tritium into t.he reaction products. [l-‘“Cl- Oleic acid (0.005 pmoles, 1.07 &i per pmole) was incubated for 30 min and 60 min at pH 5.2, 25”, with 0.12 mg of enzyme pro- tein in 0.17 ml of 3H20 enriched medium (calculated enrichment 2.6 PCi per patom of hydrogen). The reaction was stopped, the fatty acid products were extracted and separated as de- scribed above, and their aH:14C ratios were determined (Table VII, B). After 30 min of incubation, the [runs-AlO-octadecenoic acid (7% of products) had a 3H:14C ratio 54% that of the cal- culated value, based upon the incorporation of 1 atom of hy- drogen from the medium without isotope discrimination. The aH :14C ratio for the IO-hydroxyoctadecanoic acid (67 y0 of prod- ucts) was 50% that of the calculated value. After 60 min of incubation the .corresponding 3H:14C ratios had increased to 58% for the trans-A1O-octadecenoic acid (15% of products) and to 54% for the lo-hydroxyoctadecanoic acid (74oj, of products). Thus one sees an apparent 2-fold discrimination against tritium incorporation in the conversion of oleic acid to either lo-hy- drosyoctadecanoic acid or to truns-A1O-octadecenoic acid.

Addition of Hydrogen at Carbon 11--To esamine the extent of incorporat.ion of 3H from the medium at carbon 11 in the conversion of truns-A1O-oct’adecenoic acid to cisdg-octadecenoic

TABLE VIII Determi?lation of stereochemistry of hydrogen added at carbolL 9 in

Ihe reaction cis-A9-ocladecenoale --) trar&A1o-ocladece~~oale

Compound ‘H:“C

[l-14C, 9-aH]Octadecanoic acid from hydrogenation of trans-A1O-octadecenoate

Saturated fatty acids recovered from Corynebac- terium diphtheriae

Unsaturated fatty acids recovered from Corynebac- terium diphtheriae.

2.14

2.38

2.01

acid the following experiment was performed. trans-AlO-[1-14C]- Octadeccnoic acid (0.18 I.cmolc, 0.068 PCi per Imole) was in- cubated for 1 hour at pH 6, 25”, with 0.12 mg of Pseudomonas enzyme protein in 0.14 ml of 3H~0 enriched medium (cal- culated enrichment, 1.43 $Zi per patom hydrogen). The reaction was stopped, the fatty acid products were extracted and separated as described above, and their 311:14C ratios were determined (Table VII, C). The predicted 3H:14C ratio for the oleic acid (4% of products) produced by isomerization of truns-AlO-octadccenoic acid is 21 .O, based on the incorporation of 1 atom of hydrogen without isotope discrimination. The experimentally determined value was only 1.5, demonstrating a discrimination against 3I-I of greater than 9 : 1 for the addition of hydrogen at Cl1 during this 1 hour incubation. The stereo- chemistry of the added hydrogen was not determined, but is presumed to be L based upon the hydrogen abstraction data (Table VI, C). The subsequent hydration of the oleic acid formed by isomerization proceeded with incorporation of more than 7Oc/, of the theoretical amount of tritium into the lo-~- hydroxyoctadecanoic acid (570 of total products), confirming the observation of a minimal discrimination against tritium ob- served in the direct hydration of oleic acid (Table VII, 13).

EJect oj 21120 on Rate of Ilydrafion and Isomerization-The rates of hydration and isomerization of the cis-A9 double bond of oleic acid, and the rate of isomerization of truns-A1O-octadecenoic acid were measured in ‘Hz0 and in highly enriched ( >90%) *H20. For oleic acid hydration, 3.5 pmoles of [lJ4C]oleic acid were incubated with 0.6 mg of enzyme protein in a medium con- sisting of 25 ~1 of 1 M potassium phosphate buffer (pH 6.3) in 20 ml of lH20 or *HzO. The *Hz0 incubation had an over-all enrichment of 98.6 atoms Gh escess *H. Samples were removed for radiochromatographic assay at 1, 3, 8, and 20 min (Fig. 2). The hydration of oleic acid to 10.hydrosyoctadecanoic acid in the *I-1*0-enriched medium initially proceeds at about 35 the rate seen in lH20 and decreases during the course of the in- cubation. The hydration of oleic acid in ‘HZ0 is linear over 8 min, whereupon 90% of the oleic acid has been converted to lo-hydrosyoctadecanoic acid.

To study the effect of 2H20 on the rate of isomerization of cis-A9-octadecenoic acid to truns-A10-octadecenoic acid, 0.7 /*moles of [lJ4C]oleic acid were incubated as described above with 1.2 mg of enzyme protein in lH20- and *H20-enriched buffers (98.4 atoms To excess *H). The incubation was repeated at a higher level of enzyme, 5.4 mg, and phosphate buffer, 100 ~1. Samples were removed for radiochromatographic assay at 1, 3, 8, and 20 min (Fig. 3). The initial rates of isomerization of oleic acid are identical in lH20 and in 2H20, and the over-all conversion of cisdg-octadecenoic acid to [runs-AlO-octadecenoic acid is some- what greater in the 2H20 enriched medium.

The effect of 2H20 on the rate of isomerization of Iruns-AlO- octadecenoic acid to cis-A9-octadecenoic acid was complicated by the subsequent rapid hydration of the cis-A$-octadecenoic acid. We therefore measured the over-all conversion of Iruns- A1O-octadecenoic acid to IO-hydroxyoctadecanoic acid in ‘Hz0 and 2H20. tran.s-A10-[l-14C]Octadecenoic acid (4.3 Fmoles) was incubated with 5.8 mg of enzyme protein in a medium con- sisting of 100 ~1 of 1.0 M potassium phosphate buffer (pH 6.1) in 20 ml of ‘Hz0 or *H*O. For two separate incubations, the *Hz0 enrichment was 92 atoms 7. excess *H and 97.5 atoms y. excess 2H. Samples were removed for radiochromatographic assay at 4, 10, 25 and 75 min (Fig. 4). The initial rate of isom- erization and subsequent hydration of Irans-Alo-octadecenoic acid in *H20-enriched medium was less than 34 that seen in lH20.

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0 i

5

c

, 0

I I I I I 5 IO 15 20

MINUTES

20 40 60 60

MINUTES

FIG. 2 (left). Effect of 2H,0 on the rate of hydration of oleic acid to IO-hydroxyoctadecanoic acid. l , 1H20 medium; (A), 2H20 medium, 98.6 atoms y0 excess 2H. Oleic acid, 0.18 mM, was incubated at pH 6.3 with the enzyme preparation, 0.03 mg per ml.

0, ‘Hz0 medium; A., 2H20 medium, 94 atoms y, excess *H; oleic acid, 0.035 mM, was incubated at pH 6.3 with enzyme preparation, 0.27 mg per ml.

FIG. 3 (ce&er). Effect of *Hz0 on the rate of isomerization of oleic acid to tra?rsdlO-octadecenoic acid. 0, ‘Hz0 medium; A, *H*O medium, 98.4 atoms y0 excess 2H; oleic acid, 0.035 mM, incubated at pH 6.3 with enzyme preparation, 0.06 mg per ml.

FIG. 4 (right). Effect of 2HsO on the rate of isomerization and subsequent hydration of trans-Alo-octadecenoic acid to lo-hy- droxyoctadecanoic acid. l , 1H20 medium; A, *Hz0 medium, 92 atoms y. excess *H; A, 2H,0 medium, 97.5 atoms ‘j$ excess *H. !!‘rans-A1o-octadecenoic acid, 0.22 mM, was incubated at pH 6.2 with enzyme preparation, 0.29 mg per ml.

Furthermore, the over-all extent of isomerization-hydration was which is essential in the @ oxidation of unsaturated fatty acids’ greatly decreased in 2Hz0, the effect being greater in This enzyme converts cis-A3-acyl-CoA to truns-A2-acyl-CoAJ 97.5% 2Hz0 than in 92’$& 2Hz0. Since the 2Hz0 effect on the which is a substrate for enoyl-CoA hydratase. The activated rate and extent of combined isomeriaation-hydration seen here thioester form of the substrate is required, and the double bond is of considerably greater magnitude than the 2Hz0 effect on the of the trans-AZ-acyl-CoA is conjugated with the thioester car- rate and extent of direct hydration of the cis-Ag-double bond of bony1 oxygen. Davidoff and Korn (24) have described a similar oleic acid (Fig. 2) we conclude that the rate of isomerization of enzyme in mitochondria which catalyzes the reversible isom- trans-Ai0-octadecenoic acid to cis-Ag-octadecenoic acid is also erization of trans-AZ-hexadecenoyl-CoA to cis- and trans-A3- significantly reduced in 2HzO-enriched media. This is con- hexadecenoyl-CoA. This mitochondrial preparation also cat- sistent with the large discrimination against the incorporation of alyzes the hydration of trans-AZ-hexadecenoyl-CoA to 3-n-hy- tritium from water during this isomerization reaction (Table droxyhexadecanoyl-CoA. The hydration is presumably cat- VII, C). alyzed by an enzyme distinct from the isomerase.

DISCUSSION

The enzyme system we have described catalyzes the first known example of cis-trans isomerization with bond migration of an isolated double bond in an acyclic molecule. Although potentially the conversion of cis-Ag-octadecenoic acid to trans-Ai0- octadecenoic acid could occur by a hydration-dehydration mechanism, experiments detailed above have eliminated this possibility. The direct c&runs isomerization of double bonds of fatty acid derivatives is not totally without precedent. Tove and his co-workers (20-22) have extensively studied an enzyme from Butyrivibrio fibrisolvens which catalyzes the isomerization of linoleic acid (cis-AS,cis-Aiz-octadecadienoic acid) to cis-Ag, trans-Au-octadecadienoic acid. Like the system we are study- ing, the substrate for the linoleate cis-Ai2-trans-Au isomerase is the free carboxylic acid. The double bond which is isomerized is in the middle of a long hydrocarbon chain remote from any activating functional group. The enzyme, does, however, require an all cis-A9J2 diene system, and the product is stabilized by the conjugation of the cis-trans diene system (20). The effect of chain length on linoleate isomerase is remarkably sim- ilar to that seen for oleate isomerase (Table IV). The M-carbon dienoic acid, linoleic acid, is the preferred substrate, the 16-, 17-, and 19-carbon analogs being isomerized at 15 to 55% of the rate for linoleic acid. The 20-carbon analog was inactive as a substrate (21). No accompanying hydration reaction has been demonstrated for this enzyme.

Bloch and his co-workers (25) have extensively studied an enzyme from Escherichia coli which catalyzes the intercon- version of thioesters of 3-hydroxydecanoate, trans-AZ-decenoate, and cis-A3-decenoate. The mechanism has been shown to in- volve direct isomerization of trans-AZ-decenoyl thioester and cis-A3-decenoyl thioester, and also direct hydration of trans- A2-decenoyl thioester. Enzyme-bound trans-A2-decenoyl thio- ester is an intermediate in the interconversion of the &-A3 isomer and 3-hydroxydecanoyl thioester. The enzyme is very specific for the Cl0 substrate, having essentially no activity with Cs and CIZ analogs, and the activity with the biologically un- natural Cg and Cn analogs is significantly reduced. The GO acetylenic analog is isomerized to an allene conjugated with the thioester carbonyl. This conjugated allene alkylates an es- sential histidine residue at the catalytic center, irreversibly inactivating the enzyme. This is in sharp contrast to our find- ing that neither Q- nor lo-octadecynoic acid inhibited the Pseu- domonas isomerase.

Thus it should be emphasized that, although the oleate hy- dratase-isomerase has some features in common with previously described enzymes, it has the unique feature of acting upon an isolated double bond and of forming products which are not stabilized by a conjugated unsaturated system.

Stoffel et al. (23) characterized an isomerase from rat liver

Detailed knowledge of the mechanism of action of an en- zyme requires purification of the enzyme, determination of the amino acid residues present at the substrate binding site which are necessary for activity, and careful kinetic analysis. How- ever, much may be learned about enzyme mechanism by a study

1.6-

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of its substrate specificity, the stereochemistry of the trnns- fortnation of substrate to product, from the use of deuterium and tritium labeled substrates, and from stutlyiiq the reaction in 2Hz0 and 31120. Such studies do not rcquirc a homogettcous enzyme, but may be performed on rclatircly crudr cstracts.

The study of the stcrcospccificity of hytlro~en nhstraction during isomerization of the double bond furnishes considerable information about the enzytne mechanism. The key step in the stcrcospecific chemical synthesis of the appropriate tritiutn- labeled fatty acid substrates is the reduction of the tosylatc by LiAlQ. There is however a lack of total stcrrospccificity in the reduction of the optically act.& tosylatc. Hclmkamp and Rickborn (19) have rcportcd that reduction of 2-mcthancsulfon- osybutattc with LiAl*H, proceeds primarily by a nuclcophilic displacement (S,Z), but that a competing Psi-like reaction with a carbonium ion intermediate results in 20 ‘/; rawmizntion of the product. This estent of racrmization could rrsult from 600/c strrrospecific SN2 and 40%) random Ssl displaccmcnt of the sulfonyl cstcr group. The reductive rrmovnl of the sulfottyl ester should really bc termed stcreoselrctirc rather than strreo- specific. It is possible that a higher dcgrcc of stcreosrlcctivita (greater proportion of SN2 attack) might br achicwd by pcr- forming the reduction in a less polar solrrnt than trtrahydro- furan, such as dibutyl cthcr. Schrocpfrr and Bloch (10) prepared [9-GH]- and [10-n-31~]octatlecanoic acid by LiA13H4 reduction of the corresponding tosylatcs. I-pan microbiolo~icnl desaturation, the 3H:14C ratio decreased to 20 to 35% that of the starting: material, indicating that the +xthcsis had yicldcd a partially rncemic product, consisting of 65 to 80% of the dc- sired stercoisomer and 20 to 357, of thr opposite cnantiomcr. Our results (Table II) also indicate that tht, [9-t-%- and [ 1 l-L-311]-labcled fatty acids were 80 ‘i;c of the &sired sterco- isomer and 20% the opposite cnantiomc~r. This lack of total stereospecificity in the synthesis complicates the ititcrprctatioti of the cspcritnents somewhat in that the rctctttioti of 20’2 of the tritium during the enzymatic hydrog-rn al~strwtioti could bc interpreted as a lack of total stcreosprcificity in the cttzymatic reaction.

For example, in the conversion of fmns-A1O-octatlcccttoic acid to cis-Ag-octadeccnoic acid, it could bc :wgwtl that in 800; of the molecules it is the O-L hydrogen which is rcmovcd, but in 2Oc/, the 9-t) hydrogen is retnoved. If this wre the case, one would expect a loss of -2OM of the label from the trans.At0- [9-tPH]octadeccnoic acid upott cottvcrsion to cis-A”-octatlcccttoic acid. Kc found about 4 to 5% decrcasc in the 311:t4C ratio (Table II, ii), while in their study of thr drsnturation of stcaric acid to oleic acid Schroepfcr and I~locli (4) fount1 a 13 (7; tlecrcase in the 3H:14C ratio for the oleic acid drrivctl from [IO-L-~II]- stearic acid and a 7 to 11% decrease in the $11 :14C: ratio for tlic olcic acid derived from [9-t-3H]stcaric acid. If one takes itito account the pritnary kinetic isotope effect, discritninatitt~ against a clcavagc of the carbott-tritiutn bond, howwr, these data could be consistent with litnited ttonstercospcc~iticit~ of hydrogrn removal. We tnay therefore state that it is (lstremcly probable that these hydrogen abstractions occur with absolute sterco- specificity, but cannot rigorously exclude thr possibility of less than total stereospecificity.

The primary kinetic isotope effect sew for thr removal of the 9L and 11~ hydrogetts, as evidenced by the incrcascd 3H :t4C ratio of the wireacted substrates (Table II, 13 and C), indicates that the cleavage of the C-H bond in the transition state is the rate litniting step in the over-all isomcrization process. Without kinetic measurements using dcutcrium-labeled sub-

strates the magnitude of the isotope effect cannot be dcter- mined.

The strong disrriniittation against tritium in the addition of the 1 LL hydrogen in the conversion of trans.A’o-octatleccttoic acid to cis-A”-o~tatleccttoi~ acid, and the minimal discrimination against tritium in addition of the 9-L hydrogen in the rcversc reaction (Table VII) may be csplained in several ways. The hydrogctts would be cspected to be added to the substrate from a protott-donating general acid group of the enzyme, rather than added directly from hilt lyatrr. The gntcral acid group which dotiatcs the hydrogen to the 9-t, position tnust, extensively equilibrate its protons with those of the medium, as would be cspccted for the proton of a carbosyl, imitlazolc, sulfhydryl, or amino group. The protons of the gcnrral acid group which donates the hydrogen to the 1 l-t, position, however, obviously do not rquilibrate rapidly with tritiutn of the medium, since only about 7 $; of the predicted amount of tritium is incorporated into product. This may bc due to the chcmiral nature of the gcwcrnl acid group, or may bc tlue to the environment of the $eticral acid group.

The cffcct of *I&O on the rates of thr various enzymatic reac- tions (Figs. 2, 3, and 4) also is subject to several itttrrprctations. Ait enzyme may undrrgo a change in trrtiary structure whe~i transferrctl from ‘II,0 to 211L0. This change in structure may affect its rat<> of catalysis. The relative acidities of functional groups 011 the enzyme also change in 211n0. If the enzyme reaction involves trattsfrr of hydrogens which arc easily es- changeable (e.g. OH or XI-I), a change of solvent from *I-I,0 to 211s0 can have large effects on rate processes associated with the transfers. It is, hen-rwr, of interest to note that the reac- tions which proceed at a decrcascd rate in %,O, i.e. the hydration of oleic acid to 10.ltytlrosyoctndecattoic arid and the isomcriza- tion of oleic acid to fruns-A1O-octadecenoic acid, also involve estensive incorporation of tritiutn at C-9 when the incubation is carried out in medium containing %*O (Table VII, U). The isomerization of frans-AtO-octadcceiioic acid to cis-Ag-octa- dcccnoic acid is not affected by high cttricltt~~ettt of *HzO, and involves only minimal incorporation of tritiutn at C-11 when the incubation is carried out in %I20 (Table VII, C).

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Charles E. Mortimer and Walter G. Niehaus, Jr.-octadecenoic Acid : REACTION PATHWAY AND STEREOSPECIFICITY10∆-

transEnzymatic Interconversion of Oleic Acid, 10-Hydroxyoctadecanoic Acid, and

1974, 249:2833-2842.J. Biol. Chem. 

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