Studies on succinate dehydrogenase. II. On the nature of the reaction of competitive inhibitors and substrates with succinate dehydrogenase

Studies on succinate dehydrogenase. II. On the nature of the reaction of competitive inhibitors and substrates with succinate dehydrogenase

BIOCHIMICA ET BIOPHYSICA ACTA BBA 65230 STUDIES ON SUCCINATE DEHYDROGENASE. II. ON THE NATURE OF THE REACTION OF COMPETITIVE INHIBITORS AND SUBSTRAT...

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BIOCHIMICA ET BIOPHYSICA ACTA BBA

65230

STUDIES ON SUCCINATE DEHYDROGENASE. II. ON THE NATURE OF THE REACTION OF COMPETITIVE INHIBITORS AND SUBSTRATES WITH SUCCINATE DEHYDROGENASE D. V. DERVARTANIAN AND C. VEEGER* Laboratory of Biochemistry", University of Amsterdam, Amsterdam (The Netherlands) (Received February 5th, 1965)

SUMMARY

1. D- and L-malate have been found to be substrates of succinate dehydrogenase (succinate: (acceptor) oxidoreductase, EC 1.3.99.1); the oxidation product in both cases is oxaloacetate, These substrates cause a small decline of absorption in the flavin region which is followed by a time-dependent increase of absorption above 500 mf-l, corresponding to formation of the enzyme-oxaloacetate complex. The dissociation constant (K D ) of the enzyme-substrate compound is 1.4 mM for n-malate and 4.3-+9 mM for L-malate. 2. L-Chlorosuccinate is a typical substrate of succinate dehydrogenase, causing the same decline of absorbance at 460 mf-l as succinate. 3. The stereochemistry of the reaction of these substrates with the enzyme is discussed in terms of the trans-directed dehydrogenation proposed by TCHEN AND VAN MILLIGAN.

4. n-Chlorosuccinate, a competitive inhibitor of the enzyme, forms a spectrally recognizable enzyme-inhibitor compound, the spectrum differing somewhat from those obtained with other competitive inhibitors. The differences can be explained on the basis of steric hindrance from the chlorine atom. 5. GSH and cysteine cause a decline of absorbance of the enzyme, particularly at 350-410 mf-l and 470-530 mf-l. BAL has a much greater effect. 6. p~Chloromercuribellzoate causes a decline in absorbance at 380-420 mf-l and 500-550 mf-l. 7. The reaction of enzyme with thiols and p-chloromercuribenzoate suggests that both iron and sulphur (possibly the acid-labile sulphur) are intimately involved in the absorption spectrum of the enzyme.

* Present address: Department of Biochemistry, Agricultural University of Wageningen, Wageningen (The Netherlands). ** Formerly: Laboratory of Physiological Chemistry. Postal address: J. D. Meijerplein 3. Amsterdam (The Netherlands).

Bioohim, Biophys, Acta, 105 (1965) 4 24-43 6

REACTION

MECHA~lSl\l

OF

SUCCINATE DEHYDROGENASE

INTrW[)UCTl(J~

In the preceding paper of this series-, it was sh.own that addition of competitive inhibitors to succinate dehydrogenase (succinate: (acceptor) oxidoreductase, EC 1.3.99,1) results in the formation of spectrally recognizable enzyme-inhibitor complexes. The nature of these complexes and of the spectral changes brought about by the addition of substrate to the enzyme are further examined in this paper. Preliminary accounts have been given elsewhere's",

),IATERIALS

A~J)

METHODS

The preparation of succinate dehydrogenase, determination of enzyme activity and experimental procedures have been described in the first paper of this series-. Many of the materials have also been described in that paper. L-Malate dehydrogenase (EC 1.1.1.37) and t-lactate dehydrogenase (EC 1.1.1.27) were obtained from Boehringer und Sohne. z-Methylthiazole, DL-chlorosuccinic acid, DL-methylsuccinic acid, ni-bromosuccinic acid and a,tJ-dibromosuccinic acid were used as supplied by Fluka; H 20 2, trichloroacetic acid, L-cysteine and cystine were used as supplied by British Drug Houses, Ltd.; p-cWoromercuribenzoate (sodium salt) and D-malic acid (Lots M 32 13-214and M III B-205 previously shown to contain I part fumaric acid per 200 parts malic acid by paper chromatography') were used as supplied by Sigma Chemical Co.; D-malic acid (Lot J 2202, shown by paper chromatography to contain I part fumaric acid per 500 parts malic acid) was used as supplied by Mann Research Laboratories; GSSG and GSH were used as supplied by Boehringer und Sohne, i-Malic acid supplied by either Fluka (Lot 58609 73 K) or Nutritional Biochemical Corp. was found to contain appreciable amounts of succinic acid and when used as a substrate was purified by paper chromatography as previously described", n-Chlorosuccinate was prepared from t-malic acid by the method of WALDEN 4 • The product melted at 174°, [a]~ = +21.3° (c = 5.7% in water) (Literature": m.p, 176°, [aJ~ = +20.8° (c = 6.4% in water)). L-Chlorosuccinate was prepared from asparagine and NOCl by a modification of the method of TILDEN AND MARSHALL 6 and of WALDEN 6 . The product melted at 173-174°, [aJ~ = -24-7" (c = 3.5%.in water) (Literature": m.p. 174°, [aJ~ = -19·7° (c = 9.3% in water)). Oxaloacetate and pyruvate were determined as described by TAGER7. RESULTS Effects of substrates other than succinate on the spectrum of the enzyme Effect of D- and i.-malate. Fig. I shows the spectral changes brought about by the addition of D- and t-malate. There is an immediate and pronounced decrease of absorption in the flavin region, which at the wavelength of maximum decrease (460 m,u) amounts to 14.2% and 10.6 % of the original absorption for D- and t-malate, respectively. The initial decrease of absorption in the flavin region found with D- or L-malate (or DL-malate) as well as with succinate represents a hydrogen and/or electron Biochim, Biophvs, Acta, IDS (1965) 424-436

D. V. DERVARTANIAN, C. VEEGER

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400

450

500

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600

650

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Fig. 1. The effect of D- and i.-malate on the absorption spectrum of succinate dehydrogenase. Succinate dehydrogenase containing 3.5 lUg protein per ml was used. - - - , enzyme (167 units per mg protein) treated with 70 mM t-malate (chromatographically pure) minus untreated, recorded after 3.5 min; - - - - -, enzyme (r67 units per mg protein) treated with 70 mM D-malate (Mann Research) minus untreated, recorded after 3.5 min; -'-'-'-'-, enzyme (230 units per mg protein) treated with 70 mM oxaloacetate minus untreated, recorded after I min.

transfer to the flavin moiety leading to the formation of the flavin semiquinone state. The presence of the flavin semiquinone is shown by electron-spin resonance studies's". The increase of absorption in the Soo-6so-m,u. region is due to the formation of oxaloacetate by the oxidation of D- or L-malate. The oxaloacetate reacts with the enzyme to form the enzyme-oxaloacetate inhibitor cornplex-. The increase of absorption occurs slowly and reaches a maximal value with L-malatein 3-5 min and with n-malate TABLE I FORMA.TION OF OXALOACETATE FROM D- OR L-MALATE UNDER THE INFLUENCE OF SUCCINATE DEHYDROGEN ASE

n-Malate (Sigma Chemical Co., 130 mM) or r.-malate (ISO mM) was tipped from a side arm into a solution containing the enzyme (25 mumcles flavin; 133 units per mg protein) in 100 mM sodium phosphate buffer (pH 7.6). I mM EDTA in the absence of 0, at 25°, The total volume was 2.4 ml. Spectra were recorded immediately after addition of substrate and time intervals until maximal increase of absorbance in the 500-650-m!L region was observed. This was succeeded by a loss of absorbance over the entire absorption spectrum. Under identical conditions but in conventional Thunberg tubes duplicate systems were incubated for 30 min (n-malate) and IS min (L-malate), respectively. The reactions were terminated by the anaerobic (to prevent residual auto-oxidation of reduced enzyme) addition of trichloroacetic acid (to a final concentration of 5 "!o) after cooling to 0°. The contents of the reaction mixture were centrifuged for 5 min at IO 000 X gat 0° and the supernatants neutralized with IN KOH at 0°. Oxaloacetate and pyruvate determinations were then performed on these supernatants exactly as described by TAGER'. It was assumed that the sum of oxaloacetate and pyruvate found equals the amount of oxaloacetate present at the end of the experiment. Experiments were performed in duplicate with zero-time controls.

Initial decrease of absorbance ('Yo) at 460 mp Maximum increase of absorbance ('Yo) at 600 mflTime to reach maximum increase in absorbance (min) L1 Oxaloacetate (!Lmole) in duplicate experiments

Bioclum, Biopltys. Acta, 105 (19 65) 424-436

D-Malate

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17 13. 2 0.26

0.22

REACTION MECHANISM OF SUCCINATE DEHYDROGENASE

in IO-I5 min. Fig. r illustrates the formation of the enzyme-oxaloacetate complex from D- and L-malate after 3.5 min. The spectrum of the enzyme-oxaloacetate inhibitor complex is given for comparison. Table I shows a typical experiment in which the amount of oxaloacetate formed from D- and L-malate was measured. Slight activity towards D- and L-malate could be observed with ferricyanide or phenazine methosulphate-? as acceptor. Since no activity was obtained with either meso-tartrate (30mM) or D-tartrate (25 mM), it is unlikely that n-z-hydroxyacid dehydrogenase (Ee X.I.2-4) is present in the enzyme preparation. This was, in any case, hardly to be expected, since TUBBSl l has shown that z-h incubation of heart-muscle preparation at 0° with EDTA completely destroys this enzyme. EDTA was present at every stage of preparation of the succinate dehydrogenase used in our studies. The decrease of absorption in the flavin region found with D- or L-malate is somewhat less than with succinate. It is more pronounced with n-malate than Lmalate and is approximately the same under aerobic or anaerobic conditions. Fig. :2 shows that the decrease of absorption (%) at 460 mfJ- is related to the specific activity of the enzyme preparation.

a,

15

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150

SpecifIC oetivity(units/mg p-otetn)

Fig. 2. The relationship between specific activity (units per mg protein) and the decrease of absorption (%) at 460 m,t upon addition of 70 mM n- (__) or t-malate (e) to succinate dehydrogenase.

The dissociation constant (KD) of the enzyme-D-malate complex (cj. ref. r) was 1.4 mM (Fig. 3). J{D for t-malate was between 4.3 and 4.9 mM. (The former value is equal to the concentration of L-malate giving 50 % of the maximum decrease of absorption at 460 mfJ-. The latter value is after correction for the amount of enzymeoxaloacetate complex present, as determined from the absorbancy increase at 600 mfJ-. No correction was made for any enzyme-fumarate complex which might have been formed.) Effect of D- and t.-chlorosuccinate, L-Chlorosuccinate behaves as a typical substrate by causing a decrease of absorption in the flavin region upon addition to the Biochim, Biophys. Acta. 105 (1965) 424-436

D. V. DERVARTANIAN, C. VEEGER ::I.

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Fig. 3. The effect of various concentrations of n-malate on the decrease of absorbance ('Yo) of succinate dehydrogenase at 460 m,L. A preparation containing 107 units per mg protein and 4. I mg protein per m! was used in these determinations.

enzyme (maximum decrease at 460 mft amounting to 21 %) as shown in Fig. {Electron-spin resonance studies-.? showed the formation of the flavin semiquinone and confirm that t-chlorosuccinate transfers a hydrogen atom and/or electron to the flavin moiety. It has been shown by GAWRON et al,12 with particulate succinate dehydrogenase that t-chlorosuccinate is a substrate of the enzyme' and is converted to chlorofumarate. This was confirmed for the soluble enzyme, the initial rate of oxidation with a saturating amount of substrate being 46% of the rate with succinate. Also in agreement with GAWRON et al.l 2 , n-chlorosuccinate is not oxidized but acts as a competitive inhibitor of the enzyme. In agreement with this finding and the effects of competitive inhibitors studied in the previous paper-, n-chlorosuccinate causes a decrease of absorption in the flavin region (7.1 % at 450 mfl) and an increase of absorption in the 48o-540-mfl region (Fig. 5). Although the increase of absorption

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400

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500

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600

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Fig. 4. The effect of L-ch!orosuccinate on the absorption spectrum of succinate dehydrogenase. A preparation containing 238 units per mg protein and 3.8 mg protein per ml was used. - - - , treated with 67 mM t-chlorosuccmate minus untreated.

Biochim, Biophys. Acta, 105 (1965) 424-436

REACTION MECHANISM OF SUCCINATE DEHYDROGENASE

in the 48o-S40-m,u region was only one-fifth of the response with a competitive inhibitor of t he malonate type l , the same decrease of absorption in the flavin region was obtained. No electron-spin resonance signals were ob tained on addition of n-chlorosuccinate to the enzymes.". DL-Chlorosuccinate caused the same spectral and electronspin resonance changess.? as L-chlorosuccinate except that the decrease of absorption in the flavin region was slightly less (17% at 460 mp,) presumably because of the presence of the inhibitory D-stereoisomer (J{t = 3.6 mM (ref. I2)). The effect of DL+0.04

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Fig . 5. The effect of n-chlorosuccinate on the absorption spectrum of the enzyme. A preparation co ntaini ng 238 units per mg protein and 4.0 mg protein p er ml was used. - - , treated with 51 roM n -chlorosuccinate minus untreated. Fig. 6. Th e effect of DL-bromosuccinate on the absorption sp ectrum of the enzyme. Succinate dehydrogenase containing 238 un its per mg prot ein and 4.0 mg protein per ml was used . - -, treated with 20 mM DL-bromosuecinate minus untreated.

methylsuccinate on the enzyme was similar to that of DL-chlorosuccinate. DL-Bromosuccin at e (Fig. 6) caused no spectral changes characteristic of a substrate but revealed an enzyme-inhibitor complex ofthe fumarate typ e-. Since GAWRON ei al.l 2 found t hat L-bromosuccinate is a very poor substrate, this is to be exp ected. a,p'-Dibromosuccina te caused no spectral change. Presence of i.-malate dehydrogenase and fumarate hydratase in the soluble succinate dehydrogenase preparations L-Malate dehydrogenase (EC I. I.I. 37), previously reported by HELLERMAN et at.I 3 to be present in their soluble succinat e dehydrogenase preparati on, was detected in our preparation by addit ion of L-malat e and NAD+. There was an immediate increase of absorption in t.he soo-75o-m,u region as well as a slight decrease of absorpt ion in the flavin region, characteristic of the succinate dehydrogenaseoxaloacetate complex (cj. ref. I) . At the same t ime there was an increase of absorption at 340 m,u, indicating the formation of NADH. Fumarate hydratase (EC 4.2.r.2) was detected by addition of fumarate and an excess of NAD+ to the enzyme . Spectral changes characteristic of the formation of the enzyme-oxaloacetate complex were obtained as well as an increase of absorp tion at 340 m u. At a NAD+/fumarate molar ratio of 0.14, no enzyme-oxaloacetate complex formation could be observed, whereas at a ratio of 0.47 a slight amount of the complex could be detected. Maximal enzyme-oxaloacetate complex formation (and NADH formation) occurred at NAD+/fumarat e molar ratios of 4.7-470 . Biachim , Biophys, A eta, 105 (1965) 424-43 6

D. V. DERVARTANIAN,C. VERGER

430

The effect of oxidizing and reducing agents on the absorption spectrum of succiwue dehydrogenase H 20 2 (1-10 roM), GSSG (ro-go roM) or cystine (o.gmM) had no effect on the absorption spectrum of the enzyme when recorded during time intervals in which denaturation of the enzyme was negligible. Neither GSSG nor cystine affected the spectral responses to succinate or fumarate. Potassium ferricyanide was slowly

-004 350

390

430

470

510

550

Wavelength (mfJ)

Fig. 7. The effect of GSH and cysteine on the absorption spectrum of succinate dehydrogenase. Succinate dehydrogenase containing 167 units per mg protein and 4.0 mg protein per ml was used. - - - - -, treated with 77 mM GSH min'us untreated; - - - , treated with GSH followed by 42 mM fumarate minus treated with GSH; -'-'-'-, treated with 46 mM cysteine minus untreated.

reduced to potassium ferrocyanide, possibly due to oxidation of sulphhydryl groups. Fig. 7 shows that GSH causes a decline in the absorbance, particularly between 350 and 410 mf', and between 470 and 530 mf'. The GSH-treated enzyme responds normally to fumarate (Fig. 7) and to succinate. Cysteine has a smaller effect than GSH +0.02.--.....,----.----,------.--....., +0.01

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Fig. 8. The effect of z-methylthiazole at different pH values on the absorption spectrum of succinate dehydrogenase. The enzyme contained 128 units per mg protein and 4.3 mg protein per ml. - '-'-'-, treated with 170 Dll'vI z-methylthiazole at pH 8.7 mim,s untreated; - - - , as above but at pH 7.5; - - - - -, at pH 6.5.

Biocbim, Biophys. Acta, 105 (1965) 424-436

REACTION MECHANISM OF SUCCINATE DEHYDROGENASE

43 1

particularly between 470 and 530 mp,. As with GSH, cysteine does not affect the response to succinate or fumarate. Similarly, the addition of GSH or cysteine to succinate- or fumarate-treated enzyme results in the same spectral changes as if succinate and fumarate had been absent. In view of the stronger spectral effects of GSH and recalling the suggestion of CALVIN 14 that GSH (but not cysteine) contains a thiazo1ium ring under certain conditions, e.g. in slightly acid medium, the effect of addition of 2-methylthiazole was studied at three different pH values. The decrease of absorbance (Fig. 8) suggests that a part of the spectral effects of GSH could be due to an interaction of the enzyme with a thiazolium-like structure. On the other hand, there are also some clear differences between the effect of 2-methylthiazole and GSH. An alternative explanation is that these thiols react directly with the iron atom of the iron-flavin chelate at the active site. The difference in spectral response would be dependent on the affinity of the thiol for iron. It is known that thio1s react readily with metal ions-s,

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Fig. g. The effect of BAL on the abscrption spectrum of succinate dehydrogenase. Succinate dehydrogenase containing I45 units per mg protein and 4.9 mg protein per ml was used. - - , treated with BAL (120 mM) minus untreated.

Fig. 9 shows that BAL has a much greater effect than either GSH or cysteine on the absorption spectrum. There is a strong decrease of absorption in the 400-500mp, region, with a maximum decrease (36.7%) at 460 mil-, compared with r8% decrease with succinate. The increase of absorption in the 3SD-400-mfl region corresponds to 7.r% at the maximum (360 mfl). At higher wavelengths the decrease is minimum at about 530 mp,. The difference spectrum (Fig. 9) has also a slight maximum at 560 mf-l. Since BAL reacts faster with ferricyanide (or phenazine methosulphate) than with the enzyme under all conditions tried, it was not possible to test if it acts as a substrate for the enzyme. Effect of p-chloromercuribenzoate on the absorption spectrum of the enzyme p-Chloromercuribenzoate (0.3 p,mo1e per mg protein) causes a marked decrease of the absorbance, particularly in the 380-420- and 500--55o":mp, regions (Fig. IO; cj. MASSEy16). Although the spectral effects resemble somewhat those obtained with GSH, addition of succinate or fumarate to the p-chloromercuribenzoate-treated Biochim, Biophys, Acta, 105 (19 65) 424-436

D. Y. DERYARTANIAN, C. YEEGER

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Fig, 10. The effect of p-chloromercuribenzoate on succinate-reduced succinate dehydrogenase. Succinate dehydrogenase containing 167 units per mg protein and 4.0 rug protein per ml was used. - '-'- '-, untreated enzyme; - - - , plus 140 mM succinate; - - - - -, succinate followed by 0.3 flmole p-chloromercuribenzoate per mg protein. This spectrum is identical with that found by addition of p-chlorornercuribenzoate to enzyme in the absence of succinate.

enzyme has no effect on the spectrum. Similarly, the spectrum obtained by addition of p-chloromercuribenzoate to succinate dehydrogenase in the presence of succinate or fumarate is the same as that obtained with p-chloromercuribenzoate alone.

DISCUSSION

Substrates of succinate dehydrogenase (succinate, L-chlorosuccinate, L-malate, D-malate and probably L-methylsuccinate) bring about a small decline of the absorbance in the blue region of the spectrum (where flavins show their maximum absorbance) and the formation of a signal at g = z.oo in the electron-spin resonance spectrum at 77° K (refs. 3, 8). Both effects are characteristic of the formation of a flavin semiquinone. With all substrates except L-malate the electron-spin resonance signals are quite stable, although on prolonged ageing at room temperature there is a decrease in the signal at g = 2.00 and an increase of the asymmetric signal at g = I.94 and Z.01, the electron-spin resonance measurements being made at n°K. With t-malate, however, the electron-spin resonance signals disappeared after standing for only 3.5 min at room temperatures. There was also a smaller decline in the absorbance at 450-460 mfl, with L-malate than with D-malate and a more rapid formation of the band at 600 m/-l, characteristic of the enzyme-oxaloacetate complex. Although L-malate:NAD+ oxidoreductasewas present in the enzyme preparation, it could not have been responsible for the formation of oxaloacetate from Lmalate, since NAD+ was not added and there was no increase of absorption at 340 m/-l. n-z-Hydroxyacid dehydrogenase was shown to be absent. It is most likely, then, that succinate dehydrogenase was the only enzyme responsible for the oxidation of L- and n-malate by the enzyme preparation. The amount of oxaloacetate found in the an-

REACTION MECHANISM OF SUCCINATE DEHYDROGENASE

433

aerobic experiment described in Table I was about 10 times the amount of enzyme flavin. The other hydrogen acceptor was very probably fumarate, present as a trace (about 1.6 ,umoles) in the n-malate or formed (by action of fumarate hydratase) from the L-malate. The more reapid reaction with the L-malate is readily explained by the fact that more fumarate is formed from the L-malate than is originally present in the n-malate, which is of course not reacted upon by the fumarate hydratase. The smaller formation of the oxaloacetate complex with t-malate is probably due to the formation of the fumarate complex, in the presence of the appreciable amounts of fumarate derived from the L-malate. The relatively rapid formation of the enzyme-oxaloacetate complex from Lmalate as well as of the enzyme-fumarate complex also explains the instability of the electron-spin resonance signals, since these complexes do not give a signal", The formation of the tightly bound enzyme-oxaloacetate complex explains too why succinate dehydrogenase is a poor catalyst for the oxidation of L- or n-malate. The effects of n-rnalate shown in this paper cannot be ascribed to the presence of an impurity of L-malate in the n-malate used. In the first place, the KD for the n-malate (Fig. 3) is less than for t-malate. Secondly, the addition of 120 mM n-malate together with 6 mM NAD+ to the enzyme caused no increase of absorbance at 340 mft and no rapid increase in the region 500-650 mfJ. Increases at both wavelengths were found with only 2.2 mM t-malate in the presence of NAD+. HELLERMAN et al.13 have also observed the formation of oxaloacetate when Dmalate was added to a soluble preparation of succinate dehydrogenase, but they did not find any oxaloacetate formation with L-malate. The possibility that their enzyme preparation (obtained in the absence of EDTA) contained n-hydroxyacid dehydrogenase was not excluded. The finding that ri-malate is a substrate for succinate requires a discussion in the light of the conclusion of GAWRON et al.12 that the enzyme is specific for substrates with the L- configuration. FRANKE AND HOLZ1 7 showed that a number of monosubstituted derivatives of succinate could act as substrate for succinate dehydrogenase, the order of decreasing activity with halogen-substituted acids being DLchlorosuccinate, DL-bromosuccinate, DL-iodosuccinate. DL-Methylsuccinate was found to be oxidized at a slower rate than DL-chlorosuccinate. GAWRON et al.12 showed that the L- stereoisomer is the substrate in each case, and that n-chlorosuccinate and n-methylsuccinate are competitive inhibitors. The effects of t-chlorosuccinate, n-chlorosuccinate, nt-methylsuccinate and ni-bromosuccinate on the absorption spectrum of the purified enzyme, reported in the present paper, are in agreement with these findings. It should be noted, however, that, in this respect, n-chlorosuccinate was rather atypical of competitive inhibitors, in that the increase of the absorption at 480-540 rnp was only about one-fifth of that given by malonate, fumarate and related compounds, although the decrease of absorption at 460 rnp was the same as obtained with other competitive inhibitors. This difference may be due to steric hindrance caused by the chlorine atom altering the conformation of the enzyme-inhibitor complex. The stereospecificity of the enzyme for the Lenantiomorph of chlorosuccinate is also illustrated by the lack of exchange between n-chlorosuccinate and 2H20 (ref. 18) and the failures-" of n.chlorosuccinate to give an electron-spin resonance signal with the enzyme. TCHEN AND VAN MILLIGAN 19 showed that succinate dehydrogenase operates Bioohim. Biophys, Acta,

lOS

(1965) 424-43 6

D. V. DERVARTANIAN, C. VEEGER

434

by a trans mechanism, the succinate being dehydrogenated by the indiscriminate removal of the sterically indistinguishable pairs, H 1H4 or H 2H s. The mono-substituted succinate derivatives have only one trans pair which can be dehydrogenated, and the activity with L-chlorosuccinate and competitive inhibition with D-chlorosuccinate shows that only the former compound can be bound to the enzyme so that the trans hydrogen pair is in the correct configuration to be removed by the enzyme. eOOH

eOOH

The fact that n-malate, as well as L-malate, can be dehydrogenated by the enzyme is not inconsistent with the trans mechanism if (i) the hydrogen atom from the hydroxyl group can be removed as well as those bonded to carbon, or (ii) unlike the halogen and the methyl derivatives, both the D- and the L- enantiomorphs can be bound to the enzyme in a configuration suitable for abstraction of the hydrogen atoms. There are sufficient differences between the hydroxyl and the halogen or methyl groups (size, ionization, ability to fonn hydrogen bonds) to make feasible a different behaviour of malate and the other substrates. Thus, the surprising finding that n-malate is a substrate for succinate dehydrogenase can be explained on the basis of a trans mechanism for the enzyme . In this connection, we should like to draw attention to an important conclusion which can be drawn from the recent experiments of GAWRON et al.ZO• ENGLARD AND COLOWICK 21 had previously reported the isolation of deuterated succinate after addition of succinate to succinate dehydrogenase in 2HzO, under anaerobic conditions. The deuterated succinate lost half of its deuterium on oxidation by succinate dehydrogenase. However, GAWRON et al .I S found less than two deuterium atoms per molecule of succinate and have recently reporteds? that in a short-time experiment L-monodeuterosuccinate is formed. It appears then that the first hydrogen atom of a trans pair is much more rapidly exchangeable than the second. This implies that the exchange is cis-directed, although the dehydrogenation is trans, and that succinate dehydrogenase can distinguish between sterically distinguishable cis pairs, as predicted byHIRSCHMANN 22 • It is surprising that the thiols , cysteine and glutathione, which have no effect on the reactivity of the enzyme with succinate or fumarate, and the thiol-combining reagent, p-chloromercuribenzoate, which inactivates the enzyme 23 , should have rather similar effects on the spectrum. Unlike the other thiols , BAL reduces the flavin prosthetic group. The possible relationship of these effects to the iron and the acidlabile sulphur present in the enzyme in amounts equal to that of the iron 24 remains for further examination. However, they suggest that the absorption spectrum depends critically on the iron-flavin-sulphur system, so that reaction with anyone component affects the spectrum. Biochim , Biophys. A eta. 105 (1965) 424-436

REACTION MECHANISM OF SUCCINATE DEHYDROGENASE

435

There is a little doubt that both the flavin and the iron contribute to the visible absorption spectrum of succinate dehydrogenase, but the nature of the bondings involved is not known. On the one hand, flavin-free iron proteins have a spectrum similar to that of succinate dehydrogenasew-w. On the other hand, protein-free ironflavin complexes have very similar spectra2?, 28, and the absorption of the latter between 500 and 600 mt-t changes little on reduction, which is also the case with succinate dehydrogenase 29 , 30 ,1 . The possible significance of the acid-labile sulphur, which is only found in iron proteins, and of the bond between the isoalloxazine and the polypeptide chain (probably through C-8 of the isoalloxazine-t-w) must also be considered. On addition of Na 2S 20 4 flavin-free iron proteins give the asymmetric electronspin resonance signal with g.l. at 1.94 and gil at 2.01 (refs. 25, 32, 33) previously found with succinate dehydrogenase on addition of either succinate or Na 2S2 0 4 (refs. 34, 35, 8, 9). SHETHNA et at.33 have obtained strong evidence that iron is part of the structure giving the electron-spin resonance asymmetric signal. The rate of formation of this signal on addition of substrate to NADH dehydrogenase (EC 1.6.99.3) or xanthine oxidase (EC 1.2.3.2) and the rate of disappearance on addition of product are consistent with a role of the grouping responsible in the oxidoreductions catalysed by these enzymes 36 - 38 • It is probable the same is true with succinate dehydrogenase. Recent model studies have shown that, whereas the iron-flavin semiquinone'" does not show the characteristic asymmetric electron-spin resonance signal, a rather similar signal is obtained in aqueous medium at pH 5-10 in the presence of sodium nitroprusside under reducing conditionsw, However, the g.l. and gil values were reversed. It seems likely, nevertheless, that the corresponding electron-spin resonance signal in the enzyme is due to iron bound to ligands which are not yet identified, but in which possibly the acid-labile sulphur is involved. Cysteine is present in the flavin hexapeptide isolated by WANG and co-workerss-,

ACKNOWLEDGEMENTS We are indebted to Professor E. C. SLATER for his valuable advice and interest. We wish to thank Dr. J. W. CORNFORTH, Dr. P. HEMMERICH, Dr. U. K. PANDIT, Dr. J. M. TAGER and Dr. J. D. W. VAN VOORST for useful discussions. We also gratefully acknowledge the technical assistance of Mr. H. HUISMAN. This investigation was supported in part by Grant No. RG-6569 of the U.S. Public Health Service and by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). The Cary spectrophotometer was purchased from funds made available by the Rockefeller Foundation. REFERENCES I D. V. DERVARTANIAN AND 2 D. V. DERVARTANIAN AND

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