THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 243, No. 21, Issue of November 10, pp. 6684-5694, 1963

Printed in U.S.A.

The Substrate Specificity of Fumarase*

(Received for publication, June 27, 1968)

JOHN W. TEIPEL,~ G. MICHAEL HASS, AND ROBERT L. HILL

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27706

SUMMARY

The substrate specificity of fumarase has been studied by measuring the ability of the enzyme to hydrate or dehydrate derivatives of fumarate and L-malate. Fumarase was observed to hydrate fumarate and its derivatives in the order, fluorofumarate > fumarate > chlorofumarate > bromofumarate > acetylenedicarboxylate > iodofumarate > mesaconate. With the exception of fluorofumarate, water was added trans to these substrates to produce the three+?- substituted derivatives of L-malate. Fluorofumarate was hydrated to give cu-fluoromalde, which spontaneously de- composed to oxalacetate. Acetylenedicarboxylate was also hydrated by fumarase to oxalacetate. The enzyme- catalyzed dehydration of the L-malate analogues was shown for L-threo-chloromalate, (-)threo-bromomalate, L-tartrate, and threo-hydroxyaspartate.’ In addition to defining more precisely the substrate specificity of fumarase, these studies suggest that steric hindrance by the substituent groups to proper substrate binding is primarily responsible for the observed order of reactivity among the new substrates reported here. Furthermore, the monosubstituted deriva- tives of fumarate may form two nonequivalent complexes with the active site, depending upon the nature of the sub- stituent group. Finally, the region of the active site catalyz- ing the addition of hydrogen may be sterically less specific than the region catalyzing the addition of the hydroxyl group.

Earlier studies have shown that fumarase (fumarate hydratase, EC 4.2.1.2) from swine heart muscle possesses four active sites per molecule, or an average of one site per polypeptide chain subunit (1). The studies reported here have been under- taken in order to examine more closely the substrate specificity of these sites.

The kinetic parameters of the reversible hydration of fumarate to L-malate by fumarase have been well characterized (2). Fumarase was initially observed to catalyze the interconversion

* The studies reported here were supported by a research grant from the National Science Foundation.

1 Predoctoral Fellow, National Institutes of Health, 1967 to 1968.

of only fumarate and L-malate, and therefore the subst’rate specificity of the enzyme was considered to be quite rigid. Recent studies, however, have shown that fumarase will also catalyze the dehydration of L-tartrate (3) and the hydration of fluorofumarate (4). The findings reported here confirm these observations, and also show that fumarase will hydrate chloro- fumarate, bromofumarat,e, iodofumarate, mesaconate, and acetylene dicarboxylate and will dehydrate threo-chloromalate, three-bromomalate, and three-hydroxyaspartate (5). Determina- tion of certain of the kinetic parameters for these substrates has provided additional insight into the mechanism of action of fumarase and the stereochemical specificity of its active sites.

EXPERIMENTAL PROCEDURE

Substrates

The following substrates were synthesized by methods described earlier. The purity of each substrate was examined routinely by paper chromatography in the formic acid-amyl alcohol solvent system described below.

FluorofurrJaric Acid-This compound was synthesized from 1 , 1,2-trichloro-2,3,3-trifluorocyclobutane (Chemical Procure- ment Laboratories, College Point, New York) by the method of Raasch, Miegel, and Castle (6). The compound, recrystallized twice from acetonebenzene, had a melting point of 234”.

Chlorofumaric Acid-This compound was prepared according to the method of Perkin (7). A chromatographically homo- geneous compound, m.p. 191”, was obtained after two recrystalli- zations of the crude product from water.

Bromofumaric Acid-This compound was prepared by the method of Michael (8) from recrystallized isodibromosuccinic acid, which was synthesized according to the procedure of McKenzie (9). Bromofumaric acid, twice recrystallized from ethyl ether-ligroin, melted at 182-183” and appeared chroma- tographically homogeneous.

Ioclofumuric Acid-This compound, prepared as described by Thiele and Willi (lo), melted at 192-194” after two recrystalliza- tions from ethyl ether-ligroin.

Phenylfumarate-This compound was prepared as described by Almstrijm (11) and Alexander (12) from phenylsuccinic acid, obtained from the J. T. Baker Chemical Company, Philips- burg, New Jersey. Recrystallization from benzene yielded a slightly impure product (m.p. 123-124”) as judged by paper chromatography.

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Dimethylfumaric Acid-This compound was prepared from dimethylmaleic anhydride (Aldrich) by the method of Couper, Kibler, and Lutz (13). The compound melted at 240” and was chromatographically homogeneous.

nL-Erythro-chloromalic Acid-This compound was synthesized according to the procedure of Kuhn and Ebel (14) ; it melted at 142-144” after recrystallization from ethyl ether-ligroin.

DL-Erythro-bromomali Acid-This compound was also synthesized according to the method of Kuhn and Ebel (14); it melted at 136” after recrystallization from ether-ligroin.

nL-Three-chloromalic Acid-This compound was produced from cis-dicarboxyethylene oxide (14) ; it melted at 153-154” after recrystallization from ether-ligroin. The cis-dicarboxyethylene oxide was synthesized as described by Payne and Williams (15).

oz-Three-bromomalic Acid-This compound was synthesized (14) from cis-dicarboxyethylene oxide. Because crystals were not obtained, this compound was used only for qualitative studies.

Oxalacetic acid, nn-isocitric acid, nn-threo-hydroxyaspartic acid, nn-erythro-hydroxyaspartic acid, n-malic acid, L-tart.aric acid, n-tartaric acid, mesotartaric acid, mercaptosuccinic acid, and trans.aconitic acid were obtained from Calbiochem. DL-

Citamalic acid and glutaconic acid were purchased from Aldrich. Itaconic acid, n-aspartic acid, and n-aspartic acid were obtained from Sigma, and citric acid was purchased from Baker. All were used without further purification. Mesaconic acid, ob- tained from Aldrich, and acetylenedicarboxylic acid, from Calbiochem, were recrystallized once from water and twice from ether-ligroin.

Analytical Methods

Absorbance measurements were made on a Zeiss PM& II spectrophotometer equipped with a Lauda K-2/R constant temperature circulator. Optical rotation measurements were made with a Cary 60 recording spectropolarimeter. Nuclear magnetic resonance spectra were measured with a Varian A-60 recording spectrometer. The pH of all solutions was measured with a Radiometer (model PHM 22r) pH meter equipped with a type GK 2021C combined electrode.

Enzymes

Fumarase was prepared from swine heart muscle by the method of Kanarek and Hill (16). Solutions of enzyme, pre- pared as described in the preceding paper (I), had a specific activity of 33,000 f 1000 units per mg (16). Solutions of fumarase at concentrations greater than 1 mg per ml were buffered in 0.01 M Tris-acetate, pH 7.3, and solutions of the enzyme at concentrations less than 1 mg per ml were buffered in 0.01 M sodium phosphate, pH 7.3. Under these conditions fumarase lost less than 5% of its original activity over a period of 2 to 3 hours.

Malic dehydrogenase, purchased from Continental Biochemical Corporation, was solubilized by dialysis against 0.01 M Tris- acetate buffer, pH 7.3.

Kinetic Measurements

Rates of hydration and dehydration of all substrates were measured spectrophotometrically as described earlier (17). Fumarase was added to buffered solutions of a substrate at 25” and the change in absorbance was monitored at a wave length between 220 and 280 rnp in quartz cells of I- or 5.cm light path.

71 , \I I I I I I I

6 c%a

-i E 0

T I

lo

h x

w

X(mN)

FIG. 1. The ultraviolet absorption spectra of iodofumarate (O), bromofumarate (O), chlorofumarate (A), fluorofumarate (O), mesaconate (m), and oxalacetate (A) in 0.01 M Tris-acetate, pH 7.3. E is the molar extinction coefficient.

Concentrations of fumarase in the assay solution varied from 1 x 10e5 M to 2 X lo-lo M, depending upon the reactivity of the substrate. Enzyme concentration, wave length, and length of the cell were selected for use with a particular substrate such that a change in absorbance of at least 0.01 could be conveniently measured within the time period where there was apparent zero order kinetics. The extinction coefficients of the fumarate derivatives which were required for calculation of exact rates of hydration or dehydration are presented in Fig. 1.

The rate of formation of oxalacetate from L-tartrate and acetylenedicarboxylate was measured with the aid of malic dehydrogenase. In this assay, 1.0 ml of substrate, 0.1 ml of 3 x

lop3 M DPNH, and 0.1 ml of malic dehydrogenase (2000 units) were added to a quartz cuvette, and 5 ~1 of fumarase (0.37 mg per ml) were added to start the reaction. The final concentra- tions of L-tartrate ranged from 1 X lo+ M to 1 X 1O-2 M, and acetylenedicarboxylate concentrations ranged from 1 X 10F4 M

to 1 x 10e3 M. Approximately 100 set after the reaction was initiated, the decrease in absorbance was measured at 340 mp. Under these conditions the rate of oxidation of DPNH is equal to the rate of dehydration of n-tartrate or the rate of hydration of acetylenedicarboxylate.

Determination of Ti,,,, K,, K,, and K,,

Initial velocity measurements were made at six or more substrate concentrations which normally ranged from 4 to 10 times the K, of the particular substrate. Michaelis constants (Km) and maximum initial velocities (V,,,) were calculated from these measurements by the method of Lineweaver and Burk (18).

The inhibition constants of the substrate analogues, with the exception of those for mesaconate and dimethylfumarate, were determined by measuring the initial rate of dehydration of n-malate, in the presence and in the absence of the inhibitor. These measurement,s, made at concentrations of n-malate ranging from 5 x 10-S M to 1 X lOPa M, were plotted by the method of Lineweaver and Burk. K, values were calculated from these

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too- x H-$-H F

Loo- a-F-MALATE

X coo- cl

x+” Br T

Keq 6.2 12.0 3.8

H-$-OH c;l 3 0.18

kOO- OH <O.l

L-THREO-X- NH2 - MALATE

FIG. 2. Reactions catalyzed by fumarase. The structures of the monosubstituted derivatives are shown, where X denotes the functional grollp and its position of substitution. Not shown in Fig. 2, but also catalyzed by fumarase, is the hydration of acetylenedicarboxylate to oxalacetate.

curves in the usual manner. The KI values for mesaconate and dimethylfumarate were estimated with aid of the integrated rate equation (19). The high ultraviolet absorbance and low affinity of these inhibitors for fumarase prevented measurement of initial rates of reaction in the presence of inhibiting concentra- tions of the compounds. The rate of dehydration of L-malate, in the presence and in the absence of these inhibitors, therefore, was measured beyond the range of zero order kinetics, and the increase in concentration of fumarate as a function of time was evaluated from a slightly modified form of the integrated rate equation in which the term [II/K, was incorporated.

The equilibrium constants of the reactions catalyzed by fumarase were determined spectrophotometrically. Solutions of substrate (1 X lop4 M to 5 X 10m4 M) buffered in 0.01 M Tris- acetate, pH 7.3, were allowed to react with concentrations of fumarase (0.1 to 2 mg per ml) such that equilibrium was ap- proached within a few hours. The reaction was monitored between 230 and 250 rnp against a blank solution containing only fumarase at the same concentration. The equilibrium constant was calculated from the final absorbance with the appropriate extinction coefficient of the fumarate derivatives presented in Fig. 1. It was assumed that the absorbance contributed by the malate derivatives was negligible.

Paper Chromatography

Solutions (0.1 M) of the halogen derivatives of fumarate and L-malate were allowed to react with 5 mg per ml solutions of fumarase for 2 to 3 hours. The solutions were adjusted to pH 1 and filtered through a O.&J Millipore filter to remove the acid- precipitat.ed enzyme. These solutions (10 ~1) were spotted on Whatman No. 1 paper and developed by descending chroma- tography with 5 M formic acid-amyl alcohol (1 :l, v/v) for approximately 12 hours (20). The chromatograms were dried and sprayed with an ethanolic solution of bromphenol blue (0.01%). The unreacted halogen derivatives of fumarate and L-malate were chromat.ographed as controls. The purity of the substrat,es described above was also determined in this system.

Optical Rotation Measurements

Fumarase (2 ml; -5 mg per ml) was added to 1 ml of a 0.3 M

solution of substrate at pH 7.3 and the reaction was allow-cd to proceed for approximately 2 hours. The solutions were filtered (Diaflo lOOO-molecular weight exclusion ultrafilter) to remove the fumarase, and then acidified wit,h 6 N HCI to pH 2. The optical rotation of these solutions was measured at 25” + 0.1” on a Cary 60 recording spect,ropolarimeter at 589 rnp.

Samples containing only fumarase or the fumarate derivative were also processed as described above, and the rotation of these solutions was measured at 589 rnp. The background rotation measured by these control experiments did not exceed 50/c of that measured for the hydrated samples.

Nuclear Magnetic Resonance Spectra

Fumarase (10 ml; 5 mg per ml) was added to 10.0 ml of 0.2 M

mesaconate at pH 7.5. The solution was filtered with the aid of a 0.22-p Millipore filter into a sterile vial and was incubated at room temperature for 4 days. Bacterial growth was not. evident during this time. The reaction mixture was passed through a lOOO-molecular weight exclusion ultrafilter to remove the fumarase, evaporated to dryness on a rotary evaporator, and dried for 3 days over PZOS. The solid residue was dissolved in 0.5 ml of 99.7% DtO and the spectrum of this solution was measured on a Varian A-60 recording nuclear magnetic resonance spectrometer.

RESULTS

Substrate Specijicity of Fumarase

The derivatives of fumarate and malate which were found to be substrates for fumarase are shown in Fig. 2. Only fluoro- fumarate is hydrated to give an a-substituted derivative of malate. The remaining monosubstituted derivatives, chloro- fumarate, bromofumarate, iodofumarate, mesaconate, amino- fumarate, and oxalacetate, were hydrated to P-substituted derivatives of malate. In addition, it was shown that only t.he threo isomers of these P-substituted derivatives served as sub- strates for fumarase. The conformation of the active optical isomer of the malate derivat,ives was determined only for tartrate and three-chloromalate. These isomers were found to possess the same conformation about their hydroxyl carbon atoms as L-malate (21). Although the conformations of the other malate derivatives studied have not been identified, these isomers were presumed by analogy also to possess the L configuration, and have been depicted as such in Fig. 2. Although it is not indicated in the figure, fumarase also hydrates the triple bond of acetglene- dicarboxylic acid. The action of fumarase on each substrate is described in detail in the following sections.

Pluorojumarate + Hz0 e oc-Fluoromalate-The conversion of fluorofumarate to cr-fluoromalate was reported initially by Clarke, Nicklas, and Palumbo (4), but was independently observed by us (5) prior to their report. When fumarase is allowed to react with fluorofumarate there is an initial rapid decrease in absorbance at 270 rnp, followed by a slow increase in absorbance. The decrease in absorbance was attributed to the formation of a-fluoromalate, and the increase in absorbance to the spontaneous decomposition of ac-fluoromalate to form oxal- acetate and hydrofluoric acid. Evidence for this t,wo-step reaction was obtained as follows.

The liberation of hydrofluoric acid by the decomposition of

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ol-fluoromalate was monitored in a pH-stat (Radiometer). il 5.~1 aliquot of fumarase (~1 mg per ml) was added to 10 ml of 1 x 10d3 M fluorofumarate (10 pmoles) buffered with 2 x 10e3 M Tris-acetate at pH 7.3. The reaction mixture was maintained at pH 7.3 and, aft’er base uptake was complete, a total of 9.5 ,umoles of base were consumed. This indicates that approxi- mately 1 eq of acid was released per eq of fluorofumarate initially present.

Osalacetate was identified as the end product of the fumarase- catalyzed hydration of fluorofumarate with the acid of malic dehydrogenase. A rapid loss in optical density was observed at 340 rnp after the addition of 10 ~1 of fumarase (ml mg per ml) to a l-ml solution containing 1 X IOU4 M fluorofumarate, 2 X lo+ M DPNH, 10,000 units of malic dehydrogenase, and 0.01 M

Tris-acetate at pH 7.3. A 1: 1 stoichiometric relationship was observed between the concentrations of fluorofumarate hydrated and oxalacetate formed.

Studies were also performed to determine if /3-fluoromalate, the stable fluoro derivative of malate, was produced in appreciable quant,ities by the hydration of fluorofumarate. Fumarase (5 ~1; -1 mg per ml) was added to 3 ml of 3 X 10M3 M fluorofumarate buffered with 0.01 M Tris-acetate at pH 7.3, and the reaction was monitored spectrophotometrically between 265 and 280 mp. As noted above, an initial decrease in absorbance, followed by an increase in absorbance, was observed. The time required after the addition of fumarase for the rates of these opposing absorbance changes to become equal (i.e. when AA/At = 0) was measured as a function of the wave length at, which the reaction was monitored. During this assay, the concentration of fluoro- fumarate remained saturating, and thus its rate of hydration was essentially zero order (see V,,, and K, values for fluoro- fumarate). The following equations describe the expected change in absorbance with t,ime if both Q and /3 derivatives are formed.

h or-fluoromalate __f oxalacetate

ko7 Fluorofumarate

k:l p-fluoromalate (stable)

where k. and k’o are zero order rate constants and k1 is a first order rate constant.

Then

dA _=- dt

~FFU.M(~O + k’o) + kl~O*~[a-fluoromalatel

where

[cu-Fluoromalate] = 2 - ‘F 1

+FUM is the extinction coefficient of fluorofumarate at x (see Fig. l), and co-&A is the extinction coefficient of oxalacetate at X (see Fig. 1).

When

k,at = -2.3 log ‘O** - BFFUM _ _ __ kh WPUhl 1 (1) EOAA ko EOAA h

FIG. 3. Dependence of At, the time required after the addition of fumarase to a solution of fluorofumarate for dA/dt = 0, at wave length X.

where At is the time interval required for AA/At = 0 at wave length X.

If

kA ,- << 1 ICn

then

klAt = -2.3 log pAAi--M],

At was measured, as described above, at 265, 270, 275, and 280 mp, and the resulting values were plotted against log [(%4.4 - +F&/eoA&, as shown in Fig. 3. The resulting curve is in accord with the linear relationship predicted by Equa- tion 2. This result strongly suggests that the reaction proceeds by hydration of fluorofumarate to cu-fluoromalate, followed by the spontaneous conversion of the latter to oxalacetate. Further- more, the rate of formation of a-fluoromalate (7c0) must be at least 5 times greater than the rate of formation of P-fluoromalate (k’o), since a plot of

At versus tOA - EFFU.M ko WFUM --__

EOAA ko EOAA 1 A

is nonlinear, with experimental error, when Vo/ko 2 0.2. Finally, the first order rate constant for the decomposition of Lu-fluoromalate (kl) is approximately 1 x 10m2 se0, as de- termined from the slope of the curve in Fig. 3.

Chlorofumarate + Hz0 + L-Threo-chloromalate-When fu- marase (10 ~1; 1 mg per ml) was incubated with chlorofumarate (3 ml; 0.001 M in 0.01 M Tris-acetate, pH 7.3), the absorbance at 250 mp decreased progressively. The reaction of fumarase under the same conditions with a DL mixture of threo-chloro- malate produced an increase in optical density. The products of these reactions were chromatographed on paper. It may be seen from Table I that the RR values of the chlorofumarate mixture agree closely with the RF values of the three-chloromalate

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TARLE I Paper chromatography of fumarase substrates

Substrates were chromatographed on Whatman No. 1 paper before and after incubation with fumarase. Chromatograms

were developed with a 5 M formic acid-amyl alcohol mixture (l:l, v/v).

Substrate

Fumarate ..........

Malate. ............

Chlorofumarate. Three-chloromalate

Bromofumarate. Three-bromomalate

Iodofumarate.

Mesaconate

Before After incubation incubation

RF RF RF

0.81 0.33 0.82 0.32 0.34 0.83

0.79 0.50

0.80 0.50

0.81

0.86

0.46 0.81 0.50 0.79

0.47 0.50

0.55

0.50

0.82 0.79

0.82

0.85

mixture. This correspondence strongly suggests that fumarase catalyzes the interconversion of these two compounds.

The reaction of a DL mixture of erythro-chloromalate with fumarase did not produce a change in absorbance when assayed as described above. The sign of the optical rotation of threo- chloromalate, produced by the fumarase-catalyzed hydration of chlorofumarate, was measured at 589 rnp and was found to be negative. Since the conformation of ( -) threo-chloromalate at its hydroxyl carbon atom is equivalent to L-malate (21), the active chloro derivative of malate has been designated as L-

three-chloromalate. In an effort to determine whether cu-chloromalate was also

produced by the hydration of chlorofumarate, the amount of oxalacetate, which should be formed on decomposition of the ar-chloromalate, was measured. The absorbance of a reaction mixture composed of 2 ml of 1 x 1OF M chlorofumarate, 0.5 ml of 1.8 x lop3 M DPNH, 0.5 ml of malic dehydrogenase (10,000 units), and 10 pg of fumarase in 0.01 M Tris-acetate, pH 7.3, was monitored spectrophotometrically at 340 rnp before and after the addition of fumarase. Based upon the observed small decrease in absorbance, it can be calculated that the rate of formation of a-chloromalate is approximately 2yo of the rate of formation of three-chloromalate.

The equilibrium constant for hydration of chlorofumarate to L-three-chloromalate, measured spectrophotometrically, was determined to be 6.2.

Bromofumarate + Hz0 s ( -) Three-bromomalate-The reac- tion of fumarase with bromofumarate and with the three and erythro isomers of bromomalate was monitored spectrophoto- metrically under the same conditions as described for the assay of the chloro derivatives. The concentration of fumarase, however, was increased lo-fold for the assay of the bromo analogues. A decrease in absorbance with time was found for the reaction of bromofumarate with fumarase, and an increase in absorbance for the threo-bromomalate-fumarase reaction. The erythro isomer was unreactive.

The products of the reaction of bromofumarate and threo- bromomalate with fumarase were identified by paper chroma- tography. The RF values of these products are in close agree-

ment with the values for threo-bromomalate and bromofumarate, respectively, as shown in Table I. Threo-bromomalate, pro- duced by the fumarase-catalyzed hydration of bromofumarate, rotated polarized light negatively at 589 rnp. Although the absolute conformation of this optical isomer is not known, it seems probable, by analogy to (-)threo-chloromalate, that the isomer is L-three-bromomalate. ,4n equilibrium constant of 12 was calculated for the hydration of bromofumarate to threo- chloromalate.

Iodofumarate + Hz0 = Iodomalate-A decrease in optical density was observed for the reaction of 1 x 1O-3 M iodofumarate with a 1 mg per ml solution of fumarase when assayed as de- scribed above. Iodomalate was not tested as a substrate for the enzyme. The product of the reaction of iodofumarate with fumarase, although not positively identified as iodomalate, had an RF value characteristic of a halomalate when chromatographed on paper (see Table I). The hydration of iodofumarate was readily reversible as denoted by its equilibrium constant of 3.8, which was measured spectrophotometrically.

Mesaconate + Hz0 s (+)-&MethyZmalate-To 1 ml of fumarase solution (1 mg per ml) were added 100 ~1 of 1 X lop2 M mesaconate (methylfumarate). The absorbance of this reaction mixture was observed to decrease when assayed spectrophotometrically at 250 rnp. fl-Methylmalate was identified as the product of the above reaction by nuclear magnetic resonance spectroscopy. Mesaconate was incubated with fumarase until equilibrium was approached, and the products of this reaction mixture were scanned on a nuclear magnetic resonance recording spectrometer. In addition to the resonance bonds attributed to mesaconate, doublets were found at r = 8.5 ppm and r = 5.1 ppm. The doublet at r = 8.5 ppm was assigned to the 3 P-met.hyl hydrogen atoms of P-methyl- malate, and the doublet at r = 5.1 ppm to the single a-hydrogen of P-methylmalate. The intensities of the two doublets were in the expected 3 : 1 ratio. Notably absent from the spectrum was a singlet which could be attributed to the methyl hydrogen atoms of oc-methylmalate.

nn-Citramalic acid (cu-methylmalate), tested as a substrate for fumarase under assay conditions similar to those used for mesaconate, was found to be inactive. The negative results of this sensitive spectrophotometric assay eliminate the possi- bility that a small fraction of mesaconate was hydrated to (Y- methylmalate and was not detected by nuclear magnetic resonance analysis. Dimethylfumarate was also examined as a substrate and was found to be inactive. This finding is consistent with the results discussed above, since the hydration of this compound must necessarily produce an ar-methyl-substituted derivative of malate.

The equilibrium constant for the hydration of mesaconate to /3-methylmalate was determined to be 0.18 from both spectro- photometric measurements and integration of the nuclear magnetic resonance peaks for an equilibrium mixture of the two compounds.

L-Tartrak + Oxalacetate + Hz0 e Acetylenedicarboxylate + gH*O-The dehydration of Martrate to oxalacetate by fumarase was first shown by Nakamura and Ogata (3). The findings of the present investigation are in agreement with these earlier observations, but they also show that oxalacetate is formed by the fumarase-catalyzed hydration of acetylenedicarboxylate. The reaction of 100 ~1 of fumarase (~1 mg per ml) with 3 ml of 1 x 10h3 M n-tartrate or acetylenedicarboxylate produced an

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increase in optical density when assayed spectrophotometrically at 250 rnp. n-Tartrate and mesotartrate were inactive when assayed similarly. Oxalacetat,e was identified as the product of these reactions by the rate at which this compound oxidized DPNH in the presence of malic dehydrogenase. The enol tautomer of oxalacetate is believed to be the product initially formed because this tautomer, rather than the keto form, is preferentially bound by the enzyme (1). It might also be noted that the enol tautomer of oxalacetate should be predominantly in the trans-carboxyl form, because of hydrogen bonding between the cis-hydroxyl and carboxyl groups. This isomer (hydroxy- fumarate) is structurally similar to the halofumarates and mesaconate, which have been shown to be substrates for fu- marase.

The formation of oxalacetate either by the dehydration of n-tartrate or by the hydration of acetylenedicarboxylate appears to be essentially irreversible, since oxalacetate serves as a very poor substrate for the reverse reaction. The reaction of a concentrated solution of fumarase (~5 mg per ml) with 5 x lop4 M oxalacetate was monitored spectrophotometrically at 255 mp. An increase in optical density was initially observed, presumably because of the binding of the enol tautomer to the enzyme (l), followed by a very slow decrease in absorbance. The rate of decrease in absorbance (-0.01/15 min), however, was approximately equal to the rate of the spontaneous de- composition of oxalacetate into pyruvate and carbon dioxide (22)

Equilibrium constants for the dehydration of n-tartrate to oxalacetate and hydration of acetylene dicarboxylate to oxalace- tate were not quantitatively determined, since it was difficult to estimate the individual concentrations of all three compounds at equilibrium. It would appear, however, that both reactions go almost completely to oxalacetate as judged from the equilib- rium constants estimated from the V,,, values and binding constants of these compounds with the Haldane relationship. This estimate is consistent with the finding that the dehydration of (+)-tartrate to oxalacetate by the bacterial enzyme, tartaric acid dehydrase, proceeds 97 ‘+$ to completion (23).

Aminofumarate + Hz0 e Threo$-hydroxyaspartate-The reaction of 1 x low2 M threo-&hydroxyaspartate with fumarase (-1.5 mg per ml) produced an increase in optical density when assayed at 250 mp. The erythro derivative of hydroxyaspartate was inactive. The final product of the reaction of threo-/3- aspartate (threo-aminomalate) with fumarase was identified as oxalacetate with the aid of malic dehydrogenase. The formation of oxalacetate from P-hydroxyaspartate represents a deamination reaction. Fumarase, however, does not appear to catalyze this deamination reaction, since a DL mixture of aspartate was inact.ive as a substrate. It was, therefore, postulated that fumarase catalyzes the dehydration of /3-hydroxyaspartate to aminofumarate. This compound then rearranges to form iminosuccinate, which in turn spontaneously deaminates to oxalacetate. This reaction sequence is analogous to the deamina- tion mechanisms proposed for such enzymes as serine deaminase and threonine deaminase (24).

In addition to the analogues which have been described above as inactive, the following compounds did not prove to be substrates in the presence of 3 mg of fumarase per ml when assayed spectrophotometrically: citrat,e, nL-isocitrate, trans- aconitate, maleate, glutaconate, itaconate, phenylfumarate, and nn-mercaptosuccinate. Activity greater than 1% of that measured for threo-hydroxyaspartate, the least reactive analogue

classified as a substrate, was not observed for any of these compounds.

Kinetic Measurements

The initial rates of reaction of various substrates with fumarase were determined at pH 7.3 in 0.01 M Tris-acetate at 25”. The dehydration of n-tartrate and the hydration of acetylene- dicarboxylate to the enol forms of oxalacetate were measured by assay with malic dehydrogenase. The rate of equilibration between the enol (~255 = 7360) and keto (6255 N 160) tautomers (1, 25) and the preferential binding of the enol tautomer to fumarase prevented accurate ultraviolet spectrophotometric assays of the above reactions. The reaction rates of the re- maining analogues were assayed spectrophotometrically between 220 and 280 rnp. Owing to the spontaneous formation of oxalacetate from c+fluoromalate, the rate of hydration of fluorofumarate was measured at 220 rnp, where the ratio of absorbance of fluorofumarate to oxalacetate was maximum. The rate of hydration was also measured within 10 set of the addition of fumarase to the assay solution. The amount of a-fluoromalate formed by the hydration of fluorofumarate relative to the amount of oxalacetate produced by the decomposi- tion of oc-fluoromalate at time t is given by

where

[a-Fluoromalatel A =- [Oxalacetatel t-A

1 A = - (1 - e-hi) h

and lcl is the first order rate constant for the decomposition of cr-fluoromalate. When t = 10 set and kl = 1 X lo-* see-l, as measured earlier, the ratio of cr-fluoromalate to oxalacetate formed is 18.5: 1. The measured rate of decrease in optical density for the hydration of fluorofumarate is thus approximately 99% of that which would be observed if cu-fluoromalate were stable.

Michaelis constants (Km) and maximum initial velocities (V,,,) of substrates were calculated from Lineweaver-Burk plots. Inhibition of the dehydration of n-malate by both secondary substrates and nonsubstrate competitive inhibitors was also measured at pH 7.3 in 0.01 M Tris-acetate. It was possible to study competitive inhibition by the secondary substrates because the rates of reaction of these compounds with fumarase were at least 50 times slower than the dehydration of r,-malate under the conditions of the assay. The V,,,, K,, and Kr values for fumarate, n-malate, and analogues of these compounds are listed in Table II.

An examination of the kinetic parameters for the fumarate analogues reveals an interesting order of reactivity among these substrates. As indicated by the maximum initial velocity values, fumarase catalyzes the hydration of fluorofumarate approximately 3 times faster than the natural substrate, fuma- rate. The rates of hydration of the other analogues were found to proceed in the order, fumarate > chlorofumarate > bromofumarate > acetylenedicarboxylate > iodofumarate > mesaconate. The rates of hydration of the fumarate analogues were measured between pH 5 and pH 9, at substrate concentra- tions equal to 100 times the K, at pH 7.3. The order of re- activity among these analogues at each pH was the same as that

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5690 Substrate XpeciJicity of Fumarase Vol. 243, No. 21

TABLE II

Kinetic parameters of substrates and competitive inhibitors of fumarase

V ma*, K,,, and KI values were determined at pH 7.3 in 0.01 M

Tris-acetate buffer at 25”.

Compound

Fluorofumarate Fumarate........... Chlorofumarate Bromofumarate.. Acetylenedicarboxylate.. Iodofumarate Mesaconate Dimethylfumarate. Trans-aconitate.

L-Malate............. n-Thveo-chloromalate. n-Tartrate L-Three-hydroxyaspartate. L-Isocitrate Citrate.

-

?n

11

oles/?nole/mii

io,ooo

28,000" 1,300

170 125

2.6

1.4 <O.Ol <O.Ol

54,000"

340-680 55.5

O.Q-l.gb <O.Ol <O.Ol

n

K?n

x lo-5 M

2.7

0.5a 11 11 14.5 12

51

2.5" 7-14

130 750-1500b

KI

x lo-’ M

10 15

10 49

500 0.73

100 390-780b

0.13-0.26 2.2

“V mxx and K, values measured by Frieden and Alberty (26). h Measured in 0.01 M Tris-acetate at pH 9.0.

I I I I I I I

I I I I I I I 5.5 6.0 6.5 7.0 7.5 8.0 8.5

PH

FIG. 4. Dependence of Ii,,,,, on pH for chlorofumarate (0) and bromofumarate (0) iii 0.01 M Tris-acetate at 25”.

presented above. It may be noted that, with the exception of fluorofumarate, the K, and KI values of the halofumarate substrates are nearly equivalent. Thus, although t,he rate of hydration of the substrates decreases appreciably the larger the size of the halogen, the size of these substituents appears to have little effect on the affinity of the compounds for fumarase.

Mesaconate (methylfumarate) was the least reactive of the fumarate derivatives shown to be substrates. The effect on the reactivity of fumarate of substituting a second methyl group may be seen by comparing the kinetic parameters of mesaconate with those of dimethylfumarate. The disubstituted derivative,

in addition to being inactive, is bound to fumarase approsimately 10 times more poorly than the monomethylfumarate.

The kinetic constants for L-three-chloromalate, L-threo- hydroxyaspartate, and L-isocitrate were determined with racemic mixtures of these compounds as substrates or competitive inhibi- tors of the enzyme. A range of V,,,, K,, or K, values for each of these compounds was calculated from these kinetic measure- ments, assuming that the affinity of the L isomer for fumarase was equal to or greater than the affinity of the D isomer, and that only the I, isomer was a subst,rate for the enzyme. These conditions were based upon the observations that I,-malate (27) and L-tartrate (28) are bound more effectively to fumarase than their D isomers and that only the L isomers of malate, tartrate, and three-chloromalate are dehydrated by the enzyme. Fuma- rase was observed to dehydrate the malate analogues in the order, malate > three-chloromalate > tartrate > threo-hydroxy- aspartate. Although three-bromomalate was also shown to be a substrate for fumarase, its rat,e of dehydration was not quantita- tively determined since the compound could not be obtained in a pure state.

A comparison of the inhibition constants for citrate (cr-carboxy- methylmalate) and isocitrate (erythro-fl-carboxymethylmalate) reveals that the posit,ion of the carboxymethyl group greatly influences the binding of these analogues to fumarase.

An important relationship between the Michaelis and inhibi- tion constants for the derivatives of both n-malate and fumarate should be noted at this point. It may be seen that the K, and K, values determined for each substrate are in relatively close agreement. This correspondence provides strong evidence that these secondary substrates are indeed reacting with fumarase, rather than with some other enzyme contaminant.

pH Dependence

The initial rates of reaction of chlorofumarate and bromo- fumarate with fumarase were measured as a function of pH and

1 I O 5.5

I I I I I I 6.0 6.5 7.0 7.5 8.0 8.5

PH

FIG. 5. Dependence of K, on pH for chlorofumarate (0) and bromofumarat,e (0) in 0.01 M Tris-acetate at 25”.

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Issue of November 10, 1968 J. W. l’eipel, G. M. Has, and R. L. Hill 5691

the results were plotted by the method of Lineweaver and Burk. From these plots, the dependence of T/I,,,, K,, and V,,,/K, on pH for the above compounds was determined.

The variation of Vm,, with pH for chlorofumarate and bromo- fumarate, shown in Fig. 4, is very similar to that observed for fumarate (26), except that the former curves are shifted approxi- mately 1.4 pH units in the alkaline direction.

‘The dependence of Km on pH for chiorofumarate and bromo- fumarate is shown in Fig. 5. These curves, unlike the variation of K, with pH observed for fumarate, do not display a minimum at pH 6.7. They closely parallel, however, the dependence of K, on pH measured for L-malate (26).

V,,,/K, as a function of pH for chlorofumarate and bromo- fumarate is shown in Fig. 6. These curves closely resemble similar Vm/Km measurements for fumarate and L-malate (26), but are displaced about 0.2 pH unit in the alkaline direction.

DISCUSSION

Earlier investigations have contributed substantially toward our understanding of the mechanism of action of fumarase. The principal findings of these studies, relating to the stereospecificity and structural features of the active site, may be summarized as follows. (a) Both the hydrogen and hydroxyl ions of water are added to fumarate and removed from L-malate stereospecifically. This conclusion is based upon the observations that only the L isomer of malate may serve as a substrate (hydroxyl specificity) and that only the hydrogen atom erythro to the hydroxyl group of L-malate is removed during the dehydration reaction (hydrogen specificity) (29, 30). (b) Two nonidentical, dissociable groups, one in the acidic form and the other in the basic form, are required for catalysis (26). These groups are presumably in the active site and appear to be in close juxtaposition to the labile hydrogen atom and hydroxyl group of L-malate (28). (c) Two regions of positive charge appear necessary for the effective binding of substrate or competitive inhibitors to the active site (27).

The studies presented here reveal a broader degree of substrate

FIG. 6. Dependence of V,,,/K, on pH for chlorofumarate (0) and hromofumarate (0) in 0.01 M Tris-acetate at 25”.

specificity than recognized heretofore. Consequently, it is possible to gain further insight into the catalytic mechanism of fumarase by considering not only the stereospecificity of the addition (or removal) of water to the substrates shown in Fig. 2, but also the relative rates of hydration (or dehydration) of these substrates. Fluorofumarate is hydrated to give predominantly a-fluoromalate, whereas the other derivatives of fumarate are hydrated~tomgive a b-sub&ituted maiate. Furthermore, the rate of hydration of the fumarate derivatives proceeds in the order, fluorofumarate > fumarate > chlorofumarate > bromofumarate > iodofumarate > mesaconate.

In order to interpret the observed differences in the stereo- specificity of the addition of water and the rate of hydration of the fumarase substrates, it is helpful to consider the general features of the electrophilic addition of water to a double bond. Reactions of this type in free solution are believed to proceed via a carbonium ion int.ermediat,e according to the following reaction (31).

\ C=C’+ H+ 0 = -&-A- + H-’

/ \ $ OH

For compounds of the type

Y \ /

c=c / \

the direction of addition of water to the double bond as we11 as the rate of hydration of the double bond are markedly influenced by the nature of the group Y. The halogens and methyl group direct the addition of water according to Markonikoff’s rule, that is, the OH- group of water is added to the more highly substituted of the two carbon atoms of the double bond. Mechanistically, it is reasoned that these groups donate unshared p electrons to the a orbitals of the double bond and thus stabilize an intermediate of the type

-1:-I-- H +

The OH- group of water then preferentially attacks at the carbon atom bearing the substituent, to give the derivative

(32, 33). Thus, by analogy with hydration reactions in free solution, all of the derivatives of fumarate studied here should give a-substituted derivatives of malate.

The rate of hydration of derivatives of the type

\ iy c=c

/ \ may be either decreased or increased depending on the ability of the functional group to donate or withdraw u electrons from the double bond of the substituted compound. Since the addition of water across the double bond is an electrophilic reaction, Y groups which increase the u electron density of the double bond

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5692 Xubstrate SpeciJLcity of Fumarase Vol. 243, No. 21

ACETYLENEDICARBOXYLATE OXALACETATE + A coo-

d--OH 0 H+O”

-006 B

+

-H*O *

L-TARTRATE OXALACETATE

FIG. 7. Schematic representation of the active site-substrate complexes formed in conjunction with the hydration of acetylene- dicarboxylate to oxalacetate (upper) and the dehydration of L-

tartrate to oxalacetate (lower). The asymmetry of the active site is represented by four functional groups, three of which are non- equivalent (+, A, and B). L-tartrate is shown bound in the trans configuration.

will enhance the rate of addition of water whereas those which decrease the n electron density of the double bond will diminish the rate of addition. Consideration of the electronic properties of the Y groups on the fumarate derivatives studied here (32,33), indicates that the rate of hydration of these derivatives in free solution should be in the order, mesaconate > fumarate > iodofumarate > bromofumarate > chlorofumarate > fluoro- fumarate.

Clearly, neither the direction of the addition of water to the fumarate derivatives nor their rate of hydration proceeds exactly

as predicted from consideration of electrophilic addition reactions in free solution. Only fluoromalate adds water as predicted by Markonikoff’s rule, and the other halofumarate derivatives and mesaconate give rise to P-substituted malate derivatives.

Furthermore, the rate of hydration of the fumarate derivatives by fumarase is almost exactly opposite that predicted.

In order to explain the anti-Markonikoff addition of water it is proposed that, in the fumarase-catalyzed reaction, the stereo- specificity of hydration is completely determined by the orienta- tion of the substrate at the active site of the enzyme. Thus, the observation that fluorofumarate is hydrated to ar-fluoromalate whereas the other fumarate analogues are hydrated to the p- derivatives is believed to result exclusively from the fact that these two types of compounds are each bound in a different manner at the active site. A consideration of the action of fumarase on L-tartrate and acetylenedicarboxylate supports this hypothesis. As noted in “Results,” oxalacetate is produced by the hydration of acetylenedicarboxylate as well as by dehydration of L-tartrate. The postulated enzyme-substrate complexes formed in conjunction with these reactions are shown schemat- ically in Fig. 7. The active site has been represented by four functional groups, two equivalent groups labeled + and two nonequivalent groups labeled A and B. The + groups may be regarded as the two positively charged binding centers, and the

A and B groups as the two catalytic regions of the active site considered above. However, exact definition of these groups is not essential to the conclusions of this analysis. The more important function of these groups is to represent the minimum degree of asymmetry required by the active site to direct the stereospecific exchange of the hydrogen and hydroxyl ions of wat,er during the fumarase-catalyzed reactions.

If the hydrogen and hydroxyl ions of the oxalacetate formed by the hydration of acetylenedicarboxylate are related to groups A and B as shown in the upper part of Fig. 7, then the stereo- specificity of the fumarase reaction requires that the hydrogen and hydroxyl ions of L-tartrate, which are to be removed by the dehydration reaction, bear the same relationship to groups A and B. When these two conditions are satisfied, it must then follow that the hydrogen and hydroxyl groups of oxalacetate remaining after the dehydration of A-tartrate must be oriented differently with respect to the active site than these same groups of oxalacetate produced by the dehydration of acetylenedicarboxylate. The finding that oxalacetat,e may form two nonequivalent complexes with the active site thus provides a model to explain why fluoro- fumarate is hydrated to a-fluoromalate while the remaining monosubstituted derivatives of fumarate are hydrated to ,@ derivatives of malate.

It is postulated, as shown in Fig. 8, that fluorofumarate can bind in Configuration I, and upon hydration gives rise to 01- fluoromalate. The remaining analogues of fumarate can bind only in Configuration II, which results in formation of the p- malate derivatives. The ability of only fluorofumarate to bind in Con$guration I is probably related to the small size of the fluorine atom. This finding suggests that the region of the active site catalyzing the exchange of hydroxyl ion is sterically more

@ ?W@

F-FUMARATE a-F-MALATE

fJ!J .:::\@

X-FUMARATE L-THREO-X-MALATE

X=CI,Br,I,CHS,OH, NH2

FIG. 8. Schematic representation of the active site-substrate complexes formed in conjunction with the hydration of fluoro- fumarate to a-fluoromalate (upper) and the hydration of other monosubstituted fumarate derivatives, identified by their substit- uent group X, to the corresponding threo+malate derivatives (lower). The asymmetry of the active site is represented by four functional groups, three of which are nonequivalent (+, A, and B). The L-malate derivatives are shown bound in the trans configuration.

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Issue of November 10, 1968 J. W. Teipel, G. Al. Ham, and R. L. Hill 5693

restricted than the region catalyzing the exchange of hydrogen ion. The same conclusion may be drawn from the experiments which show that dimethylfumarate and citrate (ar-carboxy- methylmalate) are bound more poorly than mesaconate (methyl- fumarate) and isocitrate (erythro-carboxymethylmalate). The binding of the former two compounds necessarily introduces a bulky substituent group into the region catalyzing hydroxyl exchange, whereas the same substituent groups of the latter compounds may fit into the more spacious region near the resi- dues responsible for hydrogen exchange.

There are several possible explanations for the differences in the rate of hydration of the fumarate derivatives by fumarase. Because the observed rates are not those expected for electrophilic addition of water to a double bond in free solution, it is possible that hydration by fumarase proceeds by a mechanism different from that for hydration in free solution. However, a more likely explanation may be that the substituent groups on the substrates perturb correct enzyme-substrate interactions. This explanation is more attractive because there is an observed decrease in the rate of hydration of the fumarate derivatives as the size of the Y group increases. This relationship may also explain why trans-aconitate and phenylfumarate, which possess the bulkiest Y groups of all substrates tested, are not hydrated by fumarase. However, if steric hindrance by the Y groups is primarily responsible for the observed order of reactivity, then it is necessary to explain the fact that fluorofumarate is hydrated by fumarase at least 3 times faster than fumarate. This observation may be explained if it is assumed that fluorofumarate is bound at the active site of fumarase differently from the other substrates, as shown in Fig. 8. This mode of binding allows the OH- group to add to the carbon atom bearing the fluorine group, in accord with Markonikoff’s rule. I f hydration by fumarase proceeds via a carbonium ion intermediate similar to that formed in free solution, then the fluorine substituent may supplement the nor- mal enzymatic stabilization of the intermediate, and thus facili- tate the rate of hydration. Finally, it is also possible that the fluorine group may alter the rate of hydration of fluorofumarate by uniquely interacting with critical residues in the active site, thereby increasing the catalytic efficiency of the enzyme.

Frieden and Alberty have shown that the dependence of the kinetic parameters of fumarase on pH could be most simply explained by the following mechanism (26).

ElLil E”+‘F En+!M &+I

&a 11 KGF 11 KbEM 11 KbE 11

h ka ks F+Ene n

GE 11 K:,F i;

e E-M z En. + M

kd ks

K aEM L I1

/ &E 11

En-’ En-IF En-lM En-1

F and M represent fumarate and malate, respectively. The enzyme, E, is considered to exist in three ionized forms, En-l, En, and @+I, where En represents the active species. For this mechanism, Alberty has demonstrated that K,, and KbE, and the apparent dissociation constants, K’,BF, K’bBF, K’,,,, and K’bem may be determined from the dependency of V,,,, K,, and V,,,,,/K, on pH for either substrate (34). When k~ and kq are small relative to kl, lcz, ks, and kg then the apparent dissocia-

tion constants become equivalent to K,,,, KbZF, KaEM, and K bEM, respectively.

The variation of li,,,, K,, and TI,,/K, with pH for chloro- fumarate, bromofumarate, and n-three-chloromalate, shown in Figs. 4, 5, and 6, has been interpreted in terms of the mechanism described above and these findings have been related to similar pH studies on fumarate and n-malate. From these considera- tions the following conclusions have been drawn. (a) The dependence of V,,, on pH for chloro- and bromofumarates was very similar to that observed for fumarate, except that the curves for the halofumarate substrates were shifted 1.4 pH units in the alkaline direction. This observation suggests that the pK values of both dissociable groups in the active site are higher in the presence of bound chloro- or bromofumarate than for the enzyme-fumarate complex. The alkaline shift in these pK values is not surprising, since the binding of the more electronegative halofumarate derivative may tend to suppress hydrogen ion dissociation. (5) The K, versus pH curves for chloro- and bromo- fumarates do not display a minimum at pH 6.8, as observed with fumarate. This difference could be caused by the fact that the binding of fumarate lowers the pK value of one dissociable group in the active site, whereas the binding of chloro- and bromo- fumarates raises considerably, as noted above, the pK values of both dissociable groups. (c) Plots of the variation of Ti,,,/K, with pH for chlorofumarate and bromofumarate are very similar to the curves given for fumarate and L-malate. Since these curves are dependent only upon the dissociation constants for the free enzyme, the agreement between the functions for the second- ary substrates and those for fumarate and n-malate provides additional evidence that fumarase is responsible for the catalysis of these secondary substrates.

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3. NAKAM~RA, S., AND OGAT.L, H., J. Biol. Chem., 243,528 (1968). 4. CLARKE. D. D.. NICKLAS. W. J.. AND P~LUMBO. J.. Arch.

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John W. Teipel, G. Michael Hass and Robert L. HillThe Substrate Specificity of Fumarase

1968, 243:5684-5694.J. Biol. Chem. 

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