THE JOUHNAL OF J~OLOGICAI, CHEMISTRY 1’01. 246, No. 5, Issue of March 10, pp. 1350-1357, 1971

Printed in U.S.A.

I,-trans-2, 3-Epoxysuccilzic Acid

A NEW SUBSTRATE FOR FUMARASE”

(Received for publication, August 14, 1970)

F. ALBRIGHT~ AND G. J. SCHROEPFER, JR.

From the Division of Biochemistry, Deparfment of Chemistry ancl Chemical Engineering, University of Illinois, Urbana, Illinois 61801

SUMMARY

Fumarase from swine heart muscle has been found to catalyze the stereospecific hydration of the L isomer of frans-2,3-epoxysuccinate to yield mesotartrate. The struc- ture of the product was established by a combination of chromatographic techniques and by gas-liquid chromatog- raphy and mass spectrometry. Attempts to dissociate the fumarate-hydrating activity of fumarase from the L-trans-2,3-

epoxysuccinate-hydrating activity (including studies involv- ing partial heat inactivation and acrylamide gel electropho- resis) were unsuccessful. Fumarate acted as an inhibitor of the L-lrans-2,3-epoxysuccinate-hydrating activity of fu- marase and L-trans-2,3-epoxysuccinate inhibited the cat- alysis by fumarase of the conversion of fumarate to L-

malate.

Our finding that a soluble enzyme preparation from a pseudo- monad which catalyzes the stereospecific addition of the elements of water across the double bond of oleic acid and a number of other cis-Ag-olefinic acids (l-5) also catalyzes the stereospecific hydration of the epoxide functjion of cis- and trans-9, lo-epoxy- stearate (6, 7) led us to consider that other enzymes which cata- lyze the addition of the elements of water across a carbon-carbon double bond might also catalyze the hydration of the correspond- ing epoxides. With this possibility in mind, we directed our attention to the enzyme fumarase (fumarate hydratase, EC 4.2.1.2) which catalyzes the reversible interconversion of fu- marate and L-malate (8). Hydration of the corresponding epoxy acid (truns-2,3-epoxysuccinate) corresponding to fu- marate, would, by an analogous stereochemical course, yield mesotartrate. Moreover, the occurrence of such a process might conceivably be of physiological significance because L-

trans.2,3-epoxysuccinate is a known natural product (9-11) and mesotartrate is recognized as a potent competitive inhibitor of fumarase (12).

The purpose of this communication is to present results in- dicating that fumarase catalyzes the stereospecific hydration of

* This work was supported by Grant HE-09501 from the Na- tional Heart Institute.

f Supported by Training Grant 2G-321 from the National In- stit,utes of Health.

L-trans-2,3-epoxysuccinate to yield mesotartrate. A prelim- inary account of some of this work has been published (13).

EXPERIMENTAL PROCEDURES AND RESULTS

Materials and 1Methods-[l-14C]-Fumaric acid was purchased from Tracerlab, Inc., Waltham, Massachusetts. Fumaric acid was purchased from Calbiochem and was recrystallized from water prior to use. n-Tartaric acid, L-tartaric acid, and meso- tartaric acid were obtained from Nutritional Biochemicals. L-truns-2,3-Epoxysuccinic acid and the barium salt of the acid were generous gifts from Dr. Max Miller (Charles Pfizer and Company, Inc., Groton, Connecticut). The melting point of the acid, after recrystallization from acetonitrile, was 182-184” (literature: L-trans-2 ,3-epoxysuccinic acid, 180” (14) ; 181” (11) ; 182-184” (15)). A large quantity of L-trans.2,3-epoxysuccinate of very high purity was required for the kinetic studies described below. The acid, which was prepared from the barium salt according to the procedure described by Miller (15)) was purified by chromatography on activated silicic acid columns with a solvent mixture of petroleum ether and ether and mixtures of chloroform and ether. The purified acid, after two recrystalliza- tions from dioxane, melted at 182-184”. Gas-liquid chromato- graphic analysis of the dimethyl ester on a 3% OV-17 column showed a single component. To facilitate the detection of trace quantities of mesotartrate in this sample, the dimethyl ester was treated with bis(trimethylsilyl)trifluoroacetamide containing 1 y0 trimethylchlorosilane in chloroform and the resulting material was subjected to gas-liquid chromatographic analysis on the same column. A very small peak with a retention time similar to that of authentic bis-trimethylsilyl-dimethyl-mesotartrate was observed. Quantitative analysis, based upon prior determina- tion of detector response (peak area) to mass for dimethyl L-

trans-2,3-epoxysuccinate and bis-trimethylsilyl-dimethyl-meso- tartrate, indicated that the extent of contamination of the epoxy acid with tartrate was less than 0.01%. Fumarase from swine heart muscle was purchased from Calbiochem, Mann, and Nu- tritional Biochemicals. Fumarase stored in 2.0 M ammonium sulfate was solubilized according to the method of Robinson et al. (16). The recording of melting points and infrared and mass spectra was carried out as described previously (17). Studies involving combined gas-liquid chromatography-mass spectrometry were very generously made by Professor James A. McCloskey (Baylor University College of Medicine, Houston, Texas) with an LKB 9000 instrument with a 1% SE-30 column which m-as 1 foot in length. The column was maintained at 98”.

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Issue of March 10, 1971 F. Albright and G. J. Schroeppfer, Jr. 1351

The mass spectrometer was operated with an ionizing electron energy of 70 e.v. Perfluorokerosene was used as an internal standard to facilitate identification of the mass values. Meas- urements of optical rotation were made by means of a Durrum Jasco spectropolarimeter with cells with a l-cm light path. Methyl esters were prepared with diazomethane according to Schlenck and Gellerman (18) and Dalgliesh et al. (19). Protein concentration was measured by the method of Kanarek and Hill (20). Radioactivity was measured in a Beckman LS-250 liquid scintillation spectrometer with 2 ,bdiphenyloxazole (0.4%) in toluene-ethanol (2:l) as the scintillation fluid. Ascending paper chromatography of organic acids was carried out by a modification of the method of Reio (21) with a mixture of methyl ethyl ketone-acetone-water-formic acid (40 :2 : 6 : 1) as the de- veloping solvent. Detection of the compounds on the developed paper chromatograms was made by a modification of the method of Brown and Hall (22). RF values for mesotartaric acid, L-

trans-2,3-epoxysuccinic acid, and fumaric acid in this system were 0.27, 0.75, and 0.92, respectively. The distribution of radioactivity on developed paper chromatograms and thin layer plates was assayed as described previously (17, 23).

Preparation of [l -14C]- Dz-trans-2 , 3-Epoxysuccinic Acid- [l-14C]-nL-truns-2, 3-Epoxysuccinic acid (specific activity, 11.0 j&i per mmole) was prepared from [l-14C]-fumaric acid by a modification of the method of Payne and Williams (24). After extensive purification by silicic acid-Super Cel column chroma- tography and recrystallization from acetonitrile, the product melted at 208-210” (literature : DL-hYZ?%S-~ ,3-epoxysuccinic acid, 209” (14)). Ascending paper radiochromatographic analysis showed a single component with the same mobility as authentic L-truns-2,3-epoxysuccinic acid. The dimethyl ester (m.p. 72-73”) showed a single radioactive component (with the same mobility as that of the dimethyl ester of authentic L-trans-2,3- epoxysuccinic acid) upon thin layer chromatographic analysis on a Silica Gel G plate (solvent, benzene-ether, 1 :l). Gas- liquid chromatographic analysis of the dimethyl ester (3% OV-17 on Gas Chrom Q, 8 foot x 6 mm; column temperature, 120’) showed a single component. The infrared and mass spectra of the dimethyl ester were the same as those observed with the dimethyl ester of authentic L-trans-2,3-epoxysuccinic acid.

Another sample of [l-14C]-nL-truns-2, 3-epoxysuccinic acid of higher specific activity (5.85 mCi per mmole) was prepared by the procedure outlined above, except that the starting material and product were separated by preparative descending paper chromatography (Whatman No. 1 paper; solvent, methyl ethyl ketone-acetone-water-formic acid, 40 :35 : 6 :0.2). This sample of labeled DL-trans-2,3-epoxysuccinic acid had a radiopurity of over 99.7% as judged by ascending paper radiochromatography.

Enzymatic Conversion of trans-2 ,S-Epoxysuccinic Acid to Mesotartaric Acid: Characterization of Product-Preliminary in- cubations of [1-14C]-rm-trans-2,3-epoxysuccinic acid with fu- marase indicated the incorporation of label into a more polar component, which was separable from the substrate on ascending paper chromatography. Boiled enzyme controls were negative. The following large scale incubation was carried out to provide sufficient material for characterization of this product as meso- tartaric acid.

[l-14C]-Dr,-truns-2, 3-Epoxysuccinic acid (60 mg; 11 .O PCi per mmole) was dissolved in 0.05 M potassium phosphate buffer (3 ml) and the pH of the solution was adjusted to 7.0 by the addition of dilute sodium hydroxide. Fumarase (0.8 mg; 0.4 ml; Nutri-

tional Biochemicals) was added and the mixture was incubated at 25”. After 126 hours, an aliquot was removed for ascending paper radiochromatographic analysis. Approximately 32 y0 of the radioactivity was associated chromatographically with mesotartaric acid. Additional fumarate hydratase (0.2 mg; 0.1 ml) was added after 140 hours of incubation. The incubation was terminated after 175 hours (approximately 35% of the radio- activity was associated chromatographically with mesotartaric acid) by acidification to pH 2.0 with dilute HCI. The reaction mixture was lyophilieed and the residue was subjected to chroma- tography on an activated silicic acid column which was eluted with chloroform-butanol mixtures of increasing butanol content. The product, after purification by silicic acid column chroma- tography, was treated with diazomethane under conditions described by Schlenk and Gellerman (18) and the resulting dimethyl ester was purified by chromatography on an activated silicic acid column (18 g) with an eluting solvent of ether-pentane (80 :20). Thin layer chromatographic analysis on a Silica Gel G plate (solvent, ether-pentane, 80:20) showed a single com- ponent with the same mobility (RF 0.12) as that of the dimethyl- ester of authentic mesotartaric acid. Gas-liquid chromato- graphic analysis of the dimethyl ester of the product on a 5% diethyleneglycol succinate column (column temperature, 150”) showed a single component with the same retention time as that of the dimethyl ester of authentic mesotartaric acid. The trimethylsilyl derivative of the dimethyl ester of the product (prepared by treatment of the dimethyl ester with N,O-bis(tri- methylsilyl)acetamide) also showed a single component upon gas-liquid chromatographic analyses on columns of 5 y0 diethyl- eneglycol succinate, 3% SE-30, and 3% &F-l. The observed retention times were the same as those observed with bis-tri- methylsilyl-dimethyl-mesotartrate. The derivative of the mesotartrate was clearly distinguishable from those of D- and L-tartrate on the QF-1 column (Table I). Analysis of the trimethylsilyl derivative of the dimethyl ester of the product by combined gas-liquid chromatography-mass spectrometry indicated that the mass spectrum (Fig. 1) was identical with

TABLE I

Gas-liquid chromatographic analysis of mesolarlrate formed enzymatically from trans-2,J-epoxysuccinate

Compound

1. Bis-trimethylsilyl derivative of di- methyl ester of enzymatic prod- uct.............................

2. Bis-trimethylsilyl-dimethyl-meso- tartrate. . . .

3. Bis-trimethylsilyl-dimethyl-~-tar- trate...........................

4. Bis-trimethylsilyl-dimethyl-L-tar- trate...........................

-- DEGS* 1 SE-30C

0.63 0.63 0.37

0.63 0.63 0.37

0.41

-

Relative retention times’”

QF-Id

0.42

D Retention time relative to methyl myristate or methyl laurate. b Diethyleneglyool succinate (5%) on Chromosorb W; column

temperature, 120”; retention time of methyl myristate, 10.7 min. c SE-30 (3%) on Chromosorb W; column temperature, 150”;

retention time of methyl laurate, 16.8 min. d&F-l (3%) on Gas Chrom Q; column temperature, 115”; re-

tention time of methyl myristate, 19.2 min.

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1352 Xtereospeci$c Conversion of dram-~ ,?I-Epoxysuccinate to Mesotartrate Vol. 246, No. 5

73 .23

115 147 234

89 161 307

53 103

I.,

J

i ~ 1, 111 I .L I , I. 1 I, I, z

1 60 SO 100 120 140 160 180 200 220 240 260 280 300 320

1;1 40. 147 234 i= I61 -2 89 307 g 20 59 103

0 ” .A : :I(. “1. h’ 1”. I’ ‘. ‘. ‘, .I( 1,. .‘, 60 SO 100 120 140 160 180 200 220 240 260 280 300 320

23

11.5

2 *

m/e

FIG. 1. Mass spectra of authentic bis-trimethylsilyl-dimethyl mesotartrate (above) and the corresponding derivative of the product of the action of fumarase on DL-trans-2,%epoxysuccinate (below).

4 8 12

HOURS

FIG. 2. Time course of the formation of mesotartrate from racemic trans-2,3-epoxysuccinate. Dipotassium [l-14C]-~~-trans- 2,3-epoxysuccinate (22.3 pmoles) in water (12 ~1) was incubated with fumarase (0.13 mg; Calbiochem) in 0.01 M potassium phos- phate buffer (0.6 ml, pH 7.31) at 30”. The progress of the enzy- matic hydration was monitored by ascending paper radiochro- matographic analysis of aliquots (5 ~1) of the incubation mixture. +--+ , conversion of labeled epoxysuccinate to mesotartrate; A----A, boiled enzyme control.

that of bis-trimethylsilyl-dimethyl-mesotartrate. The mass spectrum showed a molecular ion (M) of low intensity (-0.5%; not shown in the figure) at m/e 322. That the molecular ion is mdeed at m/e 322 is indicated by the presence of a significant ion at m/e 307 (M-15) corresponding to the loss of a methyl group. The ion at m/e 263 (M-59) corresponds to the loss of a carbo- methoxy function. The ion at m/e 234 corresponds to the loss of a trimethylsilyl residue and a methyl group. The ion at m/e 161 is of diagnostic significance and corresponds to an ion +[(CHJ-Si-0-CH-COOCHa] resulting from the expected fi cleavage pattern seen in compounds of this type (see Reference 5 and references cited therein). The ions at m/e 147 and 73 are ions commonly seen (25) in the spectra of trimethylsilyl ether

derivatives and correspond to +[(CHg)-Si-0-Si(CH&] and +KW.X&l.

XtereospeciJic Nature of Enzyme-Catalyzed Conversion of km-.%?, S-Epoxysuccinic Acid to Mesotarkzric Acid-Dipotassium [I-WI-DL-trans-2,3-epoxysuccinate (22.3 Mmoles) in water (12 ~1) was incubated with fumarase (0.13 mg; Calbiochem) in 0.01 M

potassium phosphate buffer (0.6 ml; pH 7.3) at 30”. A similar incubation was performed with fumarase which had been heated at 100” for 1 hour prior to incubation. The progress of the enzymatic hydration was monitored by ascending paper radio- chromatographic analysis of aliquots (5 ~1) of the incubation mixture. The percentage of conversion of the racemic truns-2,3-

epoxysuccinate to mesotartrate as a function of time is shown in Fig. 2. The reaction proceeded to the extent of approximately 45% utilization of the added racemic substrate. The boiled enzyme control indicated no conversion of the added substrate to mesotartrate. Another incubation of the racemic, labeled trans-epoxysuccinate, carried out as described above, was termi- nated after 7 hours of incubation. Ascending paper radio- chromatographic analysis indicated approximately 42% con- version of the added racemic substrate to mesotartrate. The unreacted substrate was recovered by ascending paper chroma- tography followed by elution of the labeled acid from the paper with methanol and water. After evaporation of the methanol and water, the unreacted recovered substrate was reincubated with fumarase under the conditions described above. After 5 hours of incubation, only 0.5% conversion of the substrate to mesotartrate was observed.

The unreacted trans-2,3-epoxysuccinic acid, which was re- covered from the large scale incubation described previously and purified by silicic acid column chromatography, was dissolved in ethanol for determination of possible optical activity. The compound was found to be dextrorotatory ([cu], + 65” c, 3.45). Pure L-trans.2,3-epoxysuccinic acid is reported to have a specific rotation of -118” (c, 1.0 in ethanol) (I 5). The extent of con- version of racemic tram-2,3-epoxysuccinic acid to mesotartrate in the large scale incubation was approximately 35% (as judged by paper radiochromatographic analysis). The calculated expected value of the optical rotation is +83” based upon the literature value of the rotation for the pure L-isomer and assuming that the observed 35% conversion of racemic substrate to meso- tartrate represents 70% utilization of only one of the isomers of the added substrate. Although the observed value (f65”) is not in perfect agreement with the calculated value (+83”), this observation, coupled with the observation that the enzyme- catalyzed hydration of the racemic trans-epoxide proceeded only to the extent of -45% upon prolonged incubation of the racemic substrate, is sufficient to establish the stereospecific nature of the catalysis. Moreover, the catalysis of the hydration of the epoxide clearly involves the L-enantiomer.

Assays of Enzyme Activity-The catalysis by fumarase of the conversion of fumarate to L-malate was, in most cases, assayed by the following change in absorption at 250 rnp due to fumarate. The incubation usually contained potassium fuma- rate (1.33 InM), potassium phosphate buffer (0.01 M, pH 7.3), and fumarase (approximately 1 pg) in a total volume of 3 ml. In- cubations were carried out at 30”. The reaction was initiated by the addition of enzyme to the substrate.

The catalysis by fumarase of the conversion of m-tram-2,3- epoxysuccinic acid to mesotartaric acid was assayed by ascending paper radiochromatographic analysis of acidified (dilute HCl)

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aliquots of incubation mixtures of the enzyme and [l-WI-DL- trans-2,3-epoxysuccinic acid. The incubations were carried out at 30” in potassium phosphate buffer (0.01 M; pH 7.3). The distribution of radioactivity on the developed paper chromato- grams was analyzed by counting l-cm segments of the paper in a liquid scintillation spectrometer. The amount of product formed was calculated from the percentage of total radioactivity associated chromatographically with mesotartaric acid and the known specific activity of the substrate. The amount of meso- tartrate formed was plotted against time to estimate values of initial velocity.

Effect of Substrate Concentration on Enzyme-catalyzed Conver&n of L - trans-8, S- Epoxysuccinic Acid to Mesotartaric Acid- Fumarase (12 pg; Nutritional Biochemicals) was incubated with varying concentrations of [l-14C]-nL-trans-2,3-epoxysuccinic acid in a total volume of 0.12 ml of 0.01 M potassium phosphate buffer (pH 7.3). Values of K, and V,, (and the computed values of the corresponding standard errors) for the conversion of the L isomer of Iran-s-2,3-epoxysuccinate to mesotartrate were 5.7 X 10e4 rd(f 0.5 X 1W4 M) and 3.6 (AO.2) nmoles per min, respectively, as calculated by computer analysis of the initial velocity data with a program of Cleland (26). It was impossible to determine a turnover rate empirically with accuracy because the errors in the radioactive method used in the assay of the rate of conversion of epoxysuccinate to tartrate were excessive at the high saturating substrate concentrations required. How- ever, a turnover number at 30” (5.85 moles per min per mole) was calculated from V,,, and the reported (20) value of the molecular weight of fumarase of 194,000. The corresponding turnover number for the hydration of fumarate under the same conditions was 77,600.

Action of Di$erent Commercial Samples of Fumarase on Fuma- rate and L-trans-2, S-Epo~ysuccinate-Fumarase from Calbio- them, Nutritional Biochemicals, and Mann were solubilized as described previously, except that the buffer used was 0.01 M

potassium phosphate buffer (pH 7.5), to yield enzyme solutions which contained 0.99,0.82, and 0.54 pg of protein per ~1, respec- tively. Activity of the three samples of fumarase toward fuma- rate and DL-trans-2,3-epoxysuccinate was assayed as described previously, except that the enzymic reactions were carried out at pH 7.5. The ratios of epoxide-hydrating activity to fuma- rate-hydrating activity were essentially the same in these samples of crystalline fumarase obtained from three different commercial sources (Table II) which employed different methods for the isolation of the enzyme.’

E$ect of Partial Inactivation of Fumarase by Heat on Hydration of Fumurate and odrans-2 , S-Epozysuccinate-A solution of the enzyme fumarase (Calbiochem; 0.95 pg per ~1) in 0.01 M

potassium phosphate buffer (pH 7.3) was maintained at 30” and aliquots were removed periodically for assay of the two activities. Assay of the hydration of fumarate was carried out in triplicate as described above, except that the amount of enzyme used was 0.95 pg. The hydration of [1-WI-on-trans-2,3-epoxysuccinic acid was assayed in duplicate with 57.2 pg of the enzyme in the presence of substrate (0.1 mu) and potassium phosphate buffer (0.01 M, pH 7.3) in a total volume of 130 ~1 by the general

1 Fumarase from Calbiochem was prepared by the method of Massey (27), whereas that from Nutritional Biochemicals was prepared by the method of Frieden, Bock, and Alberty (28). In- formation concerning the method of preparation of fumarase utilized by Mann was not available.

procedure described above. A parallel and progressive decrease in both activities with time was observed (Table III).

Inhibition h Furnarate of Catalysis ky Fumarase of Conversion of o.z-transd , S-Epoxysuccinate to Mesotartrak+Varying con- centrations of [l-14C]-nn-trans-2,3-epoxysuccinate (from 0.193 to 0.772 m) in 0.01 M potassium phosphate buffer (pH 7.3) were incubated at 30” with fumarase (61.5 pg; Calbiochem) in a total volume of 60 ~1. The concentrations of fumarate studied were 0.5 and 1.5 ells (in the same buffer). All assays were carried out in duplicate. The average values of the initial velocities observed at the various substrate concentrations were sub- jected to computer analysis (26) and the values of l/V,,,, and Km/Vmx thereby obtained were used to draw straight lines for a reciprocal plot of the initial velocities as a function of substrate concentration (Fig. 3). The observed results strongly suggest that fumarate acts as a competitive inhibitor of the ensymatic hydration of n-tram-i!, 3-epoxysuccinate to yield mesotartrate.

Inhibition by L-trans-2,3-Epoxysuccinute of Catalysis by Fumarase of Conversion of Fumarate to z-Ma.?&--n-trans-2,3- Epoxysuccinic acid (1.65 g) was added to 0.01 M potassium phosphate buffer (pH 7.0) and the pH of the resulting mixture was carefully adjusted to pH 7.0 by the dropwise addition (over 2 hours) of potassium hydroxide (2 M) in 0.01 M potassium phos- phate buffer with stirring in an ice bath. The volume of the resulting mixture was adjusted to 25 ml by the addition of 0.01 M potassium phosphate buffer (pH 7.0). This buffered solution

TABLE II Activities of fumarase samples from different commercial sources

The enzyme preparations were solubilized and assayed as de- scribed previously, except that 0.01 M potassium phosphate buffer, pH 7.5, waz used in this study.

Source F sctivityD E activity* E/F

nwnOle/ min/mg

mnoles/

grotcin min/mg grokin

Calbiochem . . . . . . 0.148 2.54 17 Mann........................... 0.213 3.68 17 Nutritional Biochemicals.. . . 0.196 3.24 17

a Specific activity with respect to hydration of fumarate. b Specific activity with respect to hydration of n-trans-2,3-

epoxysuccinate.

TABLE III Partial inactivation of fumarase by heat

The enzyme solution, which was solubilized in 0.01 M potassium phosphate buffer (PH 7.3), was maintained for varying periods of time at 36” and then assayed for the two activities as described previously.

storage at 300 P activityO E activity” E/F

hrs mmle/min/mg protein moles/min/mg grotein

0 0.173 2.56 15 9 0.137 2.14 16

19.5 0.102 1.44 14 29 0.052 0.77 15

a Specific activity with respect to hydration of fumarate. b Specific activity with respect to hydration of L-bans-2,3-

epoxysuccinate.

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1354 Stereospecific Conversion of L-trans-2 ,S-Epoxysuccinate to Mesotartrate Vol. 246, No. 5

-- -4.0 0 4.0 0.0 12.0

FIG. 3. Inhibition by fumarate of the catalysis by fumarase of the conversion of n-trans-2,3-epoxysuccinate to mesotartmte. O-O, no inhibitor; A---A, 0.5 mM fumarate; +--+, 1.5 m&r fumarate; Tr, micromoles per min; L-t-ES, concentration of L-trans-2,3-epoxysuccinate (millimolar) .

(0.5 M) was used in the studies of the effect of the epoxysuccinate on fumarase as described below.

The catalysis by fumarase of the conversion of fumarate to n-malate was assayed by measuring the change in absorbance at 270 rnp with a recording spectrophotometer. Duplicate assays were performed at 30” in a total volume of 3.0 ml, which was 0.01 M with respect to potassium phosphate buffer (pH 7.0) and contained a constant amount of enzyme (Calbiochem; 1.42 pg) and varying concentrations of potassium fumarate (0.5 and 100 mrvr). The effect of three concentrations (33.3, 66.7, and 100 mM) of n-trans-2,3-epoxysuccinate was studied.2 The values of the observed initial velocities obtained from the assays with the various substrate and inhibitor concentrations were averaged and subjected to computer analysis (26) to obtain values of K,, V,,,, l/V,,,, Km/V,,, and the values of the standard error for each parameter. The values of l/V,,, and Km/V,, thereby obtained were used to draw straight lines for reciprocal plots of initial velocities as a function of substrate concentration (Fig. 4). It should be noted that, in this recipro- cal plot of velocity against substrate concentration, a common intercept on the ordinate was observed in the presence of the inhibitor. Moreover, this common intercept differs from that observed in the absence of the inhibitor. The computed values of vnulx at the various concentrations of L-bans-2, a-epoxy- succinate were essentially identical (0.26 pmole per min f 0.01, 0.26 pmole per min f 0.04, and 0.29 pmole per min =t 0.03) but significantly higher than that computed in the absence of inhibitor (0.19 f 0.01). The entire experiment was repeated with a different preparation of n-trans-2,3-epoxysuccinate and a different concentration of enzyme and the same phenomenon was observed.

The observation that Vm,, is higher in the presence of &runs- 2,3-epoxysuccinate than in its absence indicates that simple

2 Addition of the epoxide to the assay mixture does add slightly to the absorbance of the solution at 270 rng. However, no change in the absorbance at 270 rnp of a solution of n-trans-2,3-epoxy- succinate (100 mM) and fumarate hydratase (1.42 pg) could be detected during the time period and under the conditions used

10.0

i -1.0 0 1.0 2.0 3.0

x

FIG. 4. Inhibition by n-trans-2,3-epoxysuccinate of the cataly- sis by fumarase of the conversion of fumarate to L-malate. O-0, no inhibitor, A--A, 33.3 mM L-trams-2,3epoxysucci- nate; +-+, 66.7 rnM L-trans.2,3-epoxysuccinate; O-O, 100 rnM n-trans-2,3-epoxysuccinate; V, micromoles per min; F, con- centration of fumarate (millimolar).

FIG. 5. Inhibition by n-trans-2,3-epoxysuccinate of the cataly- sis by fumarase of the conversion of fumarate to n-malate. O-O, 2.5 mM fumarate; +-+, 1.5 mM fumarate; A-A, 0.83 mM fumarate; O-0, 0.67 IIIM fumarate; A-A, 0.50 mM fumarate; V, micromoles per min; I, concentration of L-Pans-2,3- epoxysuccinate (millimolar)

competitive inhibition is not occurring. Another test for com- petitive inhibition is by analysis of a plot of 1 /V, against inhibitor concentration (29). In the case of competitive inhibition, straight lines are obtained which have a common point of intersection at 1 /KI. When our data were subjected to such a graphical analysis (Fig. 5), straight lines were observed and a value of K, could be estimated (18.3 IILM). In addition, KI was also calculated by the use of the following equation (27) which holds for a competitive inhibitor.

where -l/K, is the point of intersection on the x axis of a Lineweaver-Burk reciprocal plot of velocity against substrate in the assay of the hydration of fumarate.

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concentration at various concentrations of inhibitor (I). The calculated values of K1 ranged in value from 16.6 mM to 20.7 mM with an average value of 18.8 mM, in good agreement with the value of Kr which was estimated graphically.

In searching for an explanation for the kinetic phenomena cited above, it was noted that Alberty and Bock (30) encountered what appears to be a similar case in the study of the inhibition by succinate at a single concentration (40 mM) of the catalysis by fumarase of the conversion of fumarate to malate in the presence of 0.05 M phosphate buffer. Succinate, which was known to be a relatively weak competitive inhibitor of fumarase (31), was found to inhibit the catalysis with an accompanying elevation of V max under the conditions studied. This phenom- enon was explained by the suggestion that a relatively poor inhibitor (such as succinate) could, at high concentrations, combine with the enzyme not only at the active site but also at another site to produce a catalytically active complex with different properties. To confirm and extend these findings, we carried out further studies of the inhibition of fumarase at several concentrations of succinate.

A buffered solution (0.01 1% potassium phosphate, pH 7.0) of succinate (0.5 M) was prepared as described in the case of the truns-epoxysuccinate. Duplicate assays of the enzymatic con- version of fumarate to malate were made as described in the case of the study of the effect of the trane-epoxysuccinate on the enzymic catalysis. The final assay mixtures contained fumarase (Calbiochem; 1.32 pg), 0.01 M potassium phosphate (pH 7.0), and varying concentrations of fumarate (0.5 JIIM to 2.5 InM). The effect of three concentrations of succinate, 33.3,66.7, and 100 mM, was studied. The results are summarized graphically in Fig. 6. The computed values of V,,, a t the various concentrations of succinate were essentially identical (0.30 pmole per min + 0.02, 0.37 pmole per mm f 0.06, and 0.34 pmole per min f. 0.07) but significantly different from that computed for the case in the absence of inhibitor (0.18 & 0.01). Thus, the inhibition of fumarate hydratase by L-trans-epoxysuccinate and by succinate appears to be qualitatively similar in that the presence of both of these structural analogues of fumarate causes an effect not only on K, but also on Vmax.

Since mesotartrate is known to be a potent competitive inhibitor of fumarase, consideration was given to the possibility that the observed inhibition of the enzyme by L-trarw2,3-

epoxysuccinate might be due to the presence of a small amount of mesotartrate in the sample of trans-epoxysuccinate. Possible contamination of the epoxysuccinate by tartrate was judged to be less than 0.01% on the basis of gas-liquid chromatographic analysis. At the highest level (100 mM) of L-&an+2,3-epoxy- succinate used in the inhibition studies, the maximum concen- tration of mesotartrate present in the assays would have been 10 PM. Serious consideration was also given to the effect of tartrate, generated enzymatically from trans-epoxysuccinate, on the conversion of fumarate to L-malate. Calculation of the possible amounts of mesotartrate which could have been formed enzymatically from the trans-epoxysuccinate during the studies of the effect of the L-trans-2,3-epoxysuccinate on the catalysis, by fumarase of the conversion of fumarate to n-malate indicated that the maximum concentration of tartrate expected from this source would have been considerably less than 10 PM. To clarify this situation the following experiments were performed. A buffered solution (0.01 M potassium phosphate, pH 7.0) of mesotartrate (0.1 M) was prepared as described in the case of the

. 300

./

x FIG. 6,Inhibition by succinate of the catalysis by fumarase of

the conversion of fumarate to n-malate. O-O, no inhibitor; A--A, 33.3 mM suecinate; +----+, 66.7 mM succinrtte; O-O, 100 mM succinate; V, micromoles per min; F, concen- tration of fumarate (millimolar) .

30.0

20.0

-1.0 0 1.0 2.0 3.0

,Y FIG. 7. Inhibition by mesotartrate of the catalysis by fumarase

of the conversion of fumarate to t-malate. O-0, no inhibi- tor; A---A, 0.2 mM mesotartrate; +---+, 0.4 mM mesotartrate O-O, 0.6 mM mesotartrate; V, micromoles per min; F, con- centration of fumarate (millimolar).

trans.epoxysuccinate. Duplicate assays of the enzymatic con- version of fumarate to malate were made as described in the case of the study of the effect of the truns-epoxysuccinate on the enzymic catalysis. The final assay mixtures contained fumarase (Calbiochem; 1.58 pg), 0.01 M potassium phosphate (pH 7.O), and varying concentrations of fumarate (from 0.5 to 2.5 InM). The effect of the following concentrations of mesotar- trate were studied: 0.02, 0.04, 0.06, 0.2, 0.4, and 0.6 M. No detectable effect of mesotartrate on the hydration of fumarate was noted at concentrations of 0.02, 0.04, or 0.06 mM. At the higher concentrations of tartrate, significant inhibition was observed (Fig. 7). It is noteworthy that the values of V,,, at the various concentrations of tartrate are the same as that value observed in the absence of inhibitor (V,, 0.23 pmole per min ZIZ 0.01). This is in contrast to the observation made in the case of the inhibition of the enzyme by trans-epoxysuccinate. This

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1356 Stereospee& Conversion of L-trans-2 ,S-Epoxysuccinate to Mesotartrate Vol. 246, No. 5

observation, coupled with the finding that the concentration of mesotartrate required to produce significant inhibition of the hydration of fumarate by fumarase was far in excess of the upper limit of contamination (by mesotartrate) of the sample of truns- epoxysuccinate, strongly suggests that the observed inhibition of the enzymatic hydration of fumarate by n-trans-2,3-epoxysuc- cinate was not due to inhibition by a small amount of mesotar- trate.

Fumarate- and Epoxide-hydrating Activities of Fumarase before and after Acrylamide Gel Electrophoresis-Acrylamide gel electro- phoresis was performed at pH 9.5 with a Canalco model G analytical apparatus (32) with six glass tubes (6.5 x 0.5 cm). The separating gel contained 7% acrylamide (recrystallized twice from chloroform prior to use), 0.37% bisacrylamide, and 0.07% ammonium persulfate. The stacking gel contained 2.2y0 acryla- mide, 0.56% bisacrylamide, and 0.044% riboflavin. A volume of 0.15 ml was provided for the protein sample by layering the stacking gel (0.15 ml) over 0.15 ml of a sucrose solution (40%), photopolymerizing for 30 min, and then adding the separating gel solution which was allowed to polymerize chemically in the dark for 30 min. Polymerizations were carried out at room tem- perature. The layer of sucrose was removed, the gels were placed in the apparatus, and Tris-glycine buffer (25 mM in Tris and 190 InM in glycine; pH 8.3) was added to the upper chamber. An aliquot of the fumarase solution (Calbiochem; 270 ~1; 607 pg of protein) was diluted with an equal volume of a 40% sucrose solu- tion, and aliquots (90 ~1; 101 pg of protein) were layered on each stacking gel through the buffer with a syringe. Electrophoresis was performed at 4” with a constant amperage of 3 ma per gel until the tracking dye had nearly passed through the gels (-2 hours). One gel was removed, stained with aniline black for 1 hour, and destained in 7% acetic acid at 12.5 ma for several hours. One main diffuse band with an RF of 0.22 (relative to the tracking dye) was observed which was accompanied by a trace component with an RF of 0.14. While this gel was being stained, the other five gels were removed and a section (0.3 cm) corresponding to an RF of 0.22 (previously established in pre- liminary experiments under the same conditions and subsequently confumed by the experiment cited above) was cut out from each of the five gels. These sections were mashed with a glass rod in a syringe (10 cc) and 0.01 M potassium phosphate buffer (pH 7.3; 0.5 ml) was added to extract the enzyme. The extracted protein was separated from the bulk of the gel by passage through a milli- pore filter apparatus (pore size, 1.2 p) under the pressure of the syringe plunger. The solution (~0.2 ml) was dialyzed against

TABLE IV

Fumarate- and epoxide-hydrating activities of fumarase before and after acrylamide gel electrophoresis

SEUUple F activity” E activity* E/F

mmole/min/ nmoles/min/ ?nK )rotein mg protein

Initial. . . 0.218 3.64 17

After dilution and dialysis for 3 hrs (control). . . . . 0.173 2.89 17

After electrophoresis and dialysis for3hrs . . . . . .._............. 0.188 3.08 16

n Specific activity with respect to hydration of fumarate. *Specific activity with respect to hydration of L-frans-2,3-

epoxysuccinate.

0.01 M potassium phosphate buffer (pH 7.3) for 3 hours to yield a solution containing 0.38 pg per ~1. (In this portion of the study all protein concentrations were measured by the method of Lowry et al. (33), since it was found in preliminary experiments that the method based upon ultraviolet absorption was not valid for determining the protein concentration after extraction from the gels.)

A control experiment was performed as a test for loss of ac- tivity upon dilution and dialysis of the fumarase solution. At the same time that the enzyme extracted from the gels was under- going dialysis, an aliquot (0.1 ml) of the initial fumarase solution (2.25 pg per ~1) was diluted to 0.3 ml with the phosphate buffer and dialyzed against 0.01 M potassium phosphate buffer (pH 7.3) for 3 hours to yield an enzyme solution of concentration 0.71 pg per ~1.

Assays of the hydration of fumarate were made at 270 rnp with a substrate concentration of 15 mM and a protein concentration of 0.6 pg per ml. The assay mixture was 0.01 M with respect to potassium phosphate buffer (pH 7.3). The total volume was 3.0 ml. Assays of the hydration of L-trans-2,3-epoxysuccinate were determined with a substrate concentration of 0.129 mM (23.2 nmoles of [l-14C]-nL-trans-2, 3-epoxysuccinate in a total volume of 90 ~1) and a protein concentration of 0.2 pg per ~1. All assays were performed at 30”. The measured specific activi- ties for the two reactions and the ratios of the specific activities for the two reactions are listed in Table IV. The ratio of the specific activity for the hydration of the L-trans-2,3-epoxysuc- cinate to the specific activity for the hydration of fumarate was essentially unchanged after electrophoresis.

DISCUSSION

The results reported herein indicate the catalysis by pig heart fumarase of the conversion of trans-2, 3-epoxysuecinate to meso- tartrate. The product was characterized by several chromato- graphic techniques and by combined gas-liquid chromatography- mass spectrometry. The enzymatic reaction is characterized by notable stereospecificity. The results presented herein indicate that the enzymatic reaction is stereospecific in nature and that the absolute configuration of the enantiomer acted upon by the enzyme is L.

The catalytic activity of fumarase for the hydration of L-trans- 2,3-epoxysuccinate is considerably less than that for the hy- dration of fumarate. In view of the low catalytic activity of fumarase for the hydration of the epoxide function of L-trans- 2,3-epoxysuccinate, serious consideration was given to the possi- bility that the enzymic activity was due to the presence of a small amount of another enzyme in the fumarase preparations. That this was not the case is strongly suggested by a constant ratio of epoxide-hydrating activity to fumarate-hydrating activity: (a) in samples of crystalline fumarase from three different commer- cial sources (which used different methods’ for the isolation of this enzyme) : (b) before and after partial inactivation of the en- zyme by heat; and (c) before and after acrylamide gel electro- phoresis. Moreover, fumarate was found to be a competitive inhibitor of the epoxide-hydrating activity of fumarase and L-

trans-2,3-epoxysuccinate inhibited the fumarate-hydrating ac- tivity of fumarase, although in the latter case the inhibition was not of a simple competitive nature.

It is important to note that Martin and Foster (11) have previ- ously reported the preparation of a cell-free extract of a species of Fkwobacterium (selected to grow on L-trans-epoxysuccinate as

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Issue of March 10, 1971 F. Albright and G. J. Schroepfer, Jr. 1357

a sole carbon source) which catalyzed the conversion of the epox- ide to mesotartrate. Whether this enzymic activity was due to fumarase or another enzyme was not established. More recently, Allen and Jakoby (34) have reported the isolation of an enzyme from Pseudomonas putida which catalyzed the quantitative con- version of both isomers of trans-2,3-epoxysuccinate to mesotar- trate.

The finding that fumarase catalyzes the conversion of L-trans- 2,3-epoxysuccinate to yield mesotartrate constitutes an extension of the substrate range of fumarase which, until recently (35-39, was regarded as limited to the interconversion of fumarate and L-malate. We have previously reported that a crude enzyme preparation from a pseudomonad which catalyzes the stereo- specific hydration of the As-double bond of oleic acid also cata- lyzes the stereospecific hydration of the epoxide function of &.s- and trans-9, lo-epoxystearate (6, 7). The present report con- stitutes, to our knowledge, the first description of findings which strongly suggest that a single enzyme which catalyzes the hydration of an olefin can also hydrate the corresponding epoxide.

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F. Albright and G. J. Schroepfer, Jr.-2,3-Epoxysuccinic Acid : A NEW SUBSTRATE FOR FUMARASEtransl-

1971, 246:1350-1357.J. Biol. Chem. 

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