REGULATION OF SUCCINATE DEHYDROGENASE AND TAUTOMERIZATION OF OXALOACETATE ANDREI D. VINOGRADOV, ALEXANDERB. KOTLYAR, VICTOR I. BUROV and YULIYAO. BELIKOVA Department of Biochemistry,Schoolof Biology,MoscowState University, Moscow 119899, U.S.S.R. INTRODUCTION In mammalian tissues succinate dehydrogenase catalyzes the reduction of ubiquinone by succinate, coupled with the formation of fumarate and ubiquinol. Being a component of the respiratory chain, this enzyme operates as a compulsory member of the Krebs cycle. Despite the significant progress in the elucidation of molecular and catalytic properties of succinate-ubiquinone reductase (1--4 and references cited therein), little is known about the control of the enzyme activity in mitochondria. As expected, the enzyme was shown to contain two structurally separated active sites responsible for the specific interaction with succinate and ubiquinone. The kinetics and thermodynamics of the specific ligands binding at both sites have been studied in great detail (7). Among natural constituents of the mitochondrial matrix, where the succinate binding site is located, oxaloacetate seems to be the most likely potential regulator of the enzyme activity (5-7). Two intrinsically related properties of the succinate dehydrogenase--oxaloacetate interaction, namely, the extremely high affinity of the inhibitor for the active site (Kd ~ 10-8 M) and the very low rate of enzyme-inhibitor complex dissociation (koff - 10-2 min-1) make oxaloacetate a unique possible regulator of succinate dehydrogenase. Many years ago it was shown that the addition of either malate stereoisomer to the soluble succinate dehydrogenase causes the reduction of the enzyme redox components (8). This observation unequivocally suggests that the interaction between malate and the substrate binding site of succinate dehydrogenase results in the formation of an enzyme, oxaloacetate complex. In fact, the formation of oxaloacetate tightly bound at the active site of succinate dehydrogenase treated with L-malate has been directly demonstrated (9). These results give rise to several interesting questions relevant to the control of the Krebs cycle. What is the mechanism and functional significance, if any, of malate oxidation by succinate dehydrogenase? How does succinate dehydrogenase operate in mitochondria, where the 271
A.D. VINOGRADOV,et al.
steady-state concentration of L-malate is relatively high and the rate of the oxaloacetate release from the enzyme active site is extremely low? What is the significance of other mitochondrial oxaloacetate-utilizing enzymes in the maintenance of succinate dehydrogenase in a finely balanced active/inactive state? The present article describes our experimental attempts to answer, at least partly, those questions. We will show that pure succinate-ubiquinone reductase is capable of L- or D-malate oxidation resulting in the formation of a highly inhibitory enol-oxaloacetate as an immediate reaction product. Some properties of the malate dehydrogenase activity of succinate dehydrogenase (10) point to the necessity of rapid ketonization of enol-oxaloacetate in the mitochondrial matrix. The existence of the oxaloacetate keto-enol tautomerase activity was thus predicted, and the two proteins capable of this activity were isolated from bovine heart mitochondria and purified up to an electrophoretically homogeneous state (11). One of them was identified as inactive aconitase; the other one seems to be a unique intramitochondrial oxaloacetate keto-enol tautomerase. MATERIALS AND METHODS Bovine heart mitochondria (12), Keilin-Hartree heart muscle preparation (13), and succinate-ubiquinone reductase (14) were prepared according to the published procedures. Succinate dehydrogenase (EC 184.108.40.206) and malate dehydrogenase activities of succinate-ubiquinone reductase were assayed at 25°C, pH 7.0, with the semiquinone diimine radical of N , N , N ' , N ' - t e t r a m e t h y l - p - p h e n y l e n e d i a m i n e (Wurster's blue, WB) as electron acceptor (15). The oxaloacetate keto-enol tautomerase activity (EC 220.127.116.11) was determined in a coupled malate dehydrogenase assay (enol ~ ketone direction), or by the direct spectrophotometric assay (ketone ~ enol direction) (11). The aconitase activity (EC 18.104.22.168) was determined after activation of the enzyme preparation (16). The experimental details are indicated in the legends to the Figure and Tables. RESULTS AND DISCUSSION Figure 1A demonstrates that the rapid reduction of the electron acceptor occurs when purified soluble succinate-ubiquinone reductase is added to a mixture containing D-malate and WB (curve 1). The initial burst of the activity, which was equal to - 1 0 0 nmol of malate oxidized per min per mg of protein, was followed by a rapid inactivation of the enzyme. This time-dependent decline of the activity is expected, since the only conceivable product of D-malate oxidation is oxaloacetate, which is a well-known strong inhibitor of succinate dehydrogenase (5-7). Indeed, in separate experiments using direct enzymatic assay we found that 1 mol of
REGULATION OF SUCCINATE DEHYDROGENASE
FIG. 1. Time course of malate oxidation by succinate-ubiquinone reductase. The reaction mixture contained: Mops (pH 7.0), 0.2 mM EDTA, 1 mM potassium cyanide, 0.004% Triton X-100, 1 mM o-malate, 5 mM glutamate (potassium salts) and 40 #M WB. The reaction was started by an addition of the enzyme (50/~g/ml) (indicated by arrow). (A) Curves 1 and 2, 1 mM Mops was used as a buffer; curves 3 and 4, 0.25 M'Mops was used as a buffer; curves 2 and 4, L-glutamate-oxaloacetate transaminase (140 ttg/ml) was added. (B) 1 mM Mops was used as a buffer, L-glutamate-oxaloacetate transaminase (140/~g/ml) was present; curve 1, no oxaloacetate keto-enoi tautomerase was added; curve 2,200/zg/ml of oxaloacetate kettr-cnol tautomerase-1 was added.
oxaloacetate was formed per 2 mol of the reduced one-electron acceptor under the conditions used. What seemed to be unexpected is that succinate dehydrogenase was able to turn over many times before the inhibition of the enzyme by accumulated oxaloacetate. Table 1 summarizes some catalytic properties of our preparations of succinate-ubiquinone reductase with respect to succinate and D- or L-malate dehydrogenase activities. It might be expected that the initial turnover number of succinate dehydrogenase in the malate oxidation reaction would be equal to or less than the first-order rate constant for the dissociation of the enzyme-oxaloacetate complex. However, the initial turnovers of succinate dehydrogenase in the reactions of D- or L-malate oxidation are incompatibly higher than the enzyme-oxaloacetate dissociation rate constant [17 or 8.5 min-1 and 0.02 min-1 (7), respectively]. Two possibilities might be suggested to explain such a discrepancy. The first is that the enol-form of oxaloaeetate, which seemed to be the most probable immediate product of malate oxidation at the succinate dehydrogenase active site, does not inhibit the enzyme, and the time dependent decline of the enzyme activity (Fig. 1A, curve 1) is due to the slow transformation of the non-inhibitory enol to the strongly inhibitory ketone. To check this possibility we compared the JAI~R 2~-J
2.2 1.5 O. 1
L-malate D-malate Succinate
8.5 17 1700
Turnover number* (min -1)
*Calculated on the basis of covalently bound flavin content of 6 nmol per mg (14). tDetermined by the Dixon method.
0.05 0.10 10.0
2.2 not determined
in succinate-ubiquinone reductase assay (M)t
Malonate, NEM Malonate, NEM Malonate, NEM, carboxin
TABLE 1. KINETIC PARAMETERS OF OXIDATION OF MALATE BY THE PURIFIED MITOCHONDRIAL SUCCINATE-UBIQUINONE REDUCTASE (25°C, pH 7.0, 0.25 M Mops) (10)
R E G U L A T I O N OF S U C C I N A T E D E H Y D R O G E N A S E
rates of succinate dehydrogenase inhibition by the keto- and enol-forms of oxaloacetate. It was shown that the enol-form binds to the enzyme active site three times as fast as the keto-form does and the rate of the oxaloacetate-inhibited enzyme activation is the same independently of whether the enzyme has been inhibited by any of the two tautomers (10). The second possibility, to explain the unexpectedly high initial turnover of the enzyme in the malate dehydrogenase reaction, is the existence of two kinetically distinct succinate dehydrogenase-oxaloacetate complexes. The simplest quantitative kinetic model of malate oxidation by succinate dehydrogenase, which was discussed in detail elsewhere (10), thus appeared as follows: Aox
Ks E + M
Kt E +
where M is D- or L-malate, E is succinate dehydrogenase, Aox and Are d are oxidized and reduced electron acceptors, respectively. O e and Ok are the enol and keto-forms of oxaloacetate, K s and Kp a r e the dissociation constants for E.M and E'Oe, respectively, Kt is the equilibrium constant for oxaloacetate tautomerization, Ki is the equilibrium constant for the interconversion of the intermediate E - O e complex into the dead-end and slowly dissociating complex (E.O)* and kcat is the first-order rate constant which is a measure of catalytic activity of the enzyme. According to the model, the intermediate rapidly dissociating enzymeenol-oxaloacetate complex is formed when D- or L-malate are oxidized at the active site of succinate dehydrogenase; two alternative transformations of the complex occur: the dissociation which results in the enzyme turnover, and the isomerization which produces a slowly dissociating tight dead-end enzyme-inhibitor complex (E.OA)*. An important feature of this model is that malate oxidation at the succinate dehydrogenase active site produces oxaloacetate in a strongly inhibitory and a metabolically inactive form, since all the enzymes known so far utilize or produce oxaloacetate as the keto-isomer. The keto-enol tautomerization of oxaloacetate is known to be a subject of the general acid-base catalysis, and under certain conditions the rate of interconversion may be quite low (17). Thus, it might be expected that succinate dehydrogenase interaction with L-malate in the mitochondrial matrix would result in a suicide inhibition of the
A.D. VINOGRADOV,et al.
enzyme independently of the operation of some oxaloacetate-utilizing intramitochondrial enzymes, i.e. glutamate-oxaloacetate transaminase (EC 22.214.171.124), oxaloacetate decarboxylase (EC 126.96.36.199) and citrate synthase (EC 188.8.131.52). Indeed, we found that under the conditions when the rate of keto-enol tautomerization is low (low ionic strength), the presence of glutamate-oxaloacetate transaminase and glutamate in the assay system does not prevent the rapid inactivation of succinate dehydrogenase by malate (Fig. 1A, curve 2). When the concentration of the buffer was increased (the rate of tautomeric interconversion was high), the operating transaminase prevented the suicide inhibition of the enzyme (Fig. 1A, curve 3). These results show that L-malate can be oxidized by succinate dehydrogenase. The turnover number of the enzyme in this unusual reaction is quite low, so the malate dehydrogenase activity of succinate dehydrogenase itself can hardly be of any importance in the overall metabolic flow of the Krebs cycle. However, this reaction may play an important role in the metabolic control of the cycle indirectly through the suicide inhibition of succinate dehydrogenase. Clearly, the significance of such a control would be a function of the oxaloacetate keto-enol tautomerization rate in the mitochondrial matrix. In 1968, Annet and Kosicki described an enzyme catalyzing the tautomeric interconversion of oxaloacetate (18). Although the enzyme was shown to be widely distributed in animal tissues, plants and microorganisms (18, 19), no functional role for it has been suggested. Our results on malate dehydrogenase activity of succinate dehydrogenase raised the possibility of taking a new look at the old problem. We have proposed that the mitochondrial matrix must contain factor(s) which catalyze(s) the enol-ketone interconversion of oxaloacetate. A search for such factors led us to the isolation from the mitochondrial matrix of two proteins capable of specific catalysis of oxaloacetate keto-enol tautomerization (11). Some properties of these proteins are summarized in Table 2. It is worthwhile mentioning that both proteins listed in Table 2 are quite different from oxaloacetate keto--enol tautomerase described by Annet and Kosicki (18) in respect of any characteristics. It seemed of interest to demonstrate the postulated regulatory role of oxaloacetate keto--enol tautomerase in the maintenance of succinate dehydrogenase in an active state under conditions similar to those in the mitochondrial matrix. Figure 1B demonstrates the kinetic behavior of the system reconstituted from the soluble purified succinate-ubiquinone reductase, glutamate-oxaloacetate transaminase and oxaloacetate ketoenol tautomerase in the presence of malate, glutamate and artificial electron acceptor (WB). Such a system catalyzed oxidation of malate for a long time, thus indicating that succinate dehydrogenase was maintained in an active
R E G U L A T I O N OF SUCCINATE D E H Y D R O G E N A S E
TABLE 2. SOME PROPERTIES OF O X A L O A C E T A T E TAUTOMERASES (OAT) ISOLATED FROM BOVINE H E A R T MITOCHONDRIA (I 1) OAT-1 M r (kD) Subunit structure Stability (40°C) Turnover number/rain (25°C, pH 9.0) enol --~ ketone ketone --) enol K m (~M) enol .-o ketone ketone ---) enol Specific inhibitors
37 single polypeptide no inactivation
OAT-2 80 single polypeptide unstable, tV2 ~15 min
1,600 not determined
220 not determined maleate pyrophosphate NEM
state (curve 2). When tautomerase was excluded from the reaction mixture, a rapid suicide inactivation of succinate dehydrogenase occurred, evidently because transaminase was unable to utilize the oxaloacetate produced in a metabolically inactive form. The last point we would like to discuss is the nature of the proteins catalyzing oxaloacetate keto--enol tautomerization. Two possibilities may be considered. One is that the mitochondrial matrix contains two tautomerase isoenzymes, whose only specific function is the catalysis of tautomeric interconversion of oxaloacetate. However, since the mitochondrial matrix contains several enzymes which utilize or produce oxaloacetate as substrate or product, the second possibility should also be considered, i.e. that oxaloacetate tautomerization is the side or partial reaction catalyzed by some enzymes capable of oxaloacetate binding. In our attempts to solve this dilemma, we searched for the oxaloacetate tautomerase activity of malate dehydrogenase, succinate dehydrogenase, fumarase and glutamate-oxaloacetate transaminase. None of those showed any tautomerase activity. A significant similarity of molecular masses of tautomerase-2 (80 kD) and of aconitase (82 kD) (20) caught our attention. It also seemed remarkable that aconitase catalyzes the transformation of that part of the citrate molecule which originated from oxaloacetate (21). A comparison of some molecular and catalytic properties of tautomerase-2 and of aconitase (Table 3) unambiguously demonstrated that the larger enzyme isolated as oxaloacetate tautomerase-2 can be identified as inactive aconitase. Whether aconitase and/or oxaloacetate tautomerase-2 should be considered as bifunctional enzymes, or that the oxaloacetate keto-enol tautomerase activity of aconitase is just a previously unknown partial property of the enzyme, remains to be answered. Whatever the answer would be the ability of inactive aconitase to catalyze tautomeric interconversion of oxaloacetate, which undoubtedly involves the proton
A. D. VINOGRADOV, et al. TABLE 3. COMPARISON OF OXALOACETATE TAUTOMERASE-2 AND ACONITASE OAT-2 (11)
Mr (kD) Subunit structure Catalytic properties
Spectral properties Prosthetic groups
80+5 single polypeptide catalyzes tautomerization of oxaloacetate catalyzes aconitase reaction after treatment with Fe2+ very sensitive to fluorocitrate K i D,L-isocitrate = 23 ~M (tautomerase reaction) K i D,L-isocitrate = 73/zM (aconitase reaction) Maximum at 418 nm Non-heme Fe (2 atom/mol) Acid-labile S (2 atom/mol)
Aconitase (16, 20, 21) 83 single polypeptide inactive as prepared catalyzes aconitase reaction after treatment with Fe2+ very sensitive to fluorocitrate K m D,L-isocitrate = 140 ~M Maximum at 418 nm Non-heine Fe (3 atom/mol) Acid-labile S (4 atom/mol)
abstraction reaction, and would shed more light on the mechanism of the aconitase reaction (22). Our attempts to identify oxaloacetate keto-enol tautomerase-1 with any known intramitochondrial enzyme were unsuccessful. Thus, at the moment we hold an opinion that the smaller protein listed in Table 2 (37 kD) is the unique intramitochondrial oxaloacetate keto-enol tautomerase. Whatever the nature of the proteins described in this report, the importance of enzymatically catalyzed oxaloacetate tautomerization in the regulation of the Krebs cycle has been demonstrated. SUMMARY
Highly purified succinate-ubiquinone reductase catalyzes the oxidation of L- or D-malate with a K m and initial Vmax equal to - 10-3 M and 100 nmol/min/mg of protein, respectively. The malate dehydrogenase activity of succinate dehydrogenase rapidly decreases regardless of the presence of glutamate plus glutamate-oxaloacetate transaminase. The inhibitor trapping system, however, prevents the inactivation of succinate dehydrogenase under the conditions when the rate of tautomeric o x a l o a c e t a t e e n o l ~--* o x a l o a c e t a t e k e t o n e i n t e r c o n v e r s i o n is high. T h e s e results suggest t h a t e n o l o x a l o a c e t a t e is an i m m e d i a t e p r o d u c t o f m a l a t e o x i d a t i o n at t h e s u c c i n a t e d e h y d r o g e n a s e active site. T w o p r o t e i n s (Mr 37 a n d 80 k D ) which c a t a l y z e the o x a l o a c e t a t e t a u t o m e r a s e r e a c t i o n w e r e isolated from the mitochondrial matrix. Some physico-chemical and kinetic p r o p e r t i e s o f t h e s e e n z y m e s w e r e c h a r a c t e r i z e d . T h e l a r g e r p r o t e i n was i d e n tified as inactive a c o n i t a s e . T h e s y s t e m c o n t a i n i n g s u c c i n a t e d e h y d r o g e n a s e , L - m a l a t e , g l u t a m a t e plus t r a n s a m i n a s e a n d o x a l o a c e t a t e t a u t o m e r a s e was
REGULATION OF SUCCINATE D E H Y D R O G E N A S E
reconstituted. Such a system is capable of oxidizing malate to aspartate without rapid inactivation of succinate dehydrogenase. Taken together, the data obtained emphasize a significant role of enzymatic oxaloacetate tautomerization in the control of the succinate dehydrogenase activity in the mitochondrial matrix.
REFERENCES 1. Y. HATEF1 and D. L. ST1GALL, Succinate dehydrogenases, pp. 222-256 in The Enzymes, Vol. 13, (P. D. BOYER, ed.), Academic Press, Inc., New York (1976). 2. A. D. VINOGRADOV, Succinate-ubiquinone reductase of the respiratory chain, Biochimiya (U.S.S.R.) 51, 1944-1973 (1663-1688 in English translation) (1986). 3. L. HEDERSTEDT and L. RUTBERG, Succinate dehydrogenase - - a comparative review, Microbiol. Rev. 45, 542-555 (1981). 4. T. P. SINGER, E. B. KEARNEY and M. GUTMAN, Regulation of succinate dehydrogenase in mitochondria, pp. 271-301 in Biochemical Regulatory Mechanisms in Eukaryotic Cells (E. KUN and S. GRISOLIA, eds.), John Wiley, Inc. (1972). 5. N. B. DAS, Studies on inhibition of the succinic and lactic-malic dehydrogenases, Biochem. J. 31, 1116-1123 (1937). 6. A. B. P A R D E E and V. R. POTTER, Inhibition of succinic dehydrogenase by oxaloacetate, J. Biol. Chem. 176, 1075-1084 (1948). 7. A. B. KOTLYAR and A. D. VINOGRADOV, Interaction of the membrane-bound succinate dehydrogenase with substrate and competitive inhibitors, Biochim. Biophys. Acta 784, 24-34 (1984). 8. D . V . DERVARTANIAN and C. VEEGER, Studies on succinate dehydrogenase II. On the nature of the reaction of competitive inhibitors and substrates with succinate dehydrogenase, Biochim. Biophys. Acta 105,424-436 (1965). 9. B. A. C. ACKRELL, E. B. KEARNEY and M. MAYR, Role of oxaioacetate in the regulation of mammalian succinate dehydrogenase, J. Biol. Chem. 249, 2021-2027 (1974). 10. Y. O. BELIKOVA, A. B. KOTLYAR and A. D. VINOGRADOV, Oxidation of malate by the mitochondrial succinate-ubiquinone reductase, Biochim. Biophys. Acta 936, in press (1988). 11. Y. O. BEL1KOVA, V. I. BUROV and A. D. VINOGRADOV, Isolation and properties of oxaloacetate keto-enol tautomerases from bovine heart mitochondria, Biochim. Biophys. Acta 936, in press (1988). 12. F.L. CRANE, J. L. GLENN and D. E. GREEN, Studies on the electron transfer system. IV. The electron transfer particles, Biochim. Biophys. Acta 22, 475-493 (1956). 13. A . D . V I N O G R A D O V and T. E. KING, The Keilin-Hartree heart muscle preparation, Methods in Enzymology 55, 118-127 (1979). 14. P. R. TUSHURASHVILI, E. V. GAVRIKOVA, A. N. LEDENEV and A. D. VINOGRADOV, Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase, Biochim. Biophys. Acta 809, 145-159 (1985). 15. A . D . VINOGRADOV, V. G. GRIVENNIKOVA and E. V. GAVRIKOVA, Studies on the succinate dehydrogenating system. I. Kinetics of the succinate dehydrogenase interaction with a semiquindiimine radical of N,N,N',N'-tetramethyl-p-phenylenediamine, Biochim. Biophys. Acta 545, 141-154 (1979). 16. J. J. VILLAFRANCA and A. S. MILDVAN, The mechanism of aconitase action. I. Preparation, physical properties of the enzyme, and activation by iron (II), J. Biol. Chem. 246, 772-779 (1971). 17. M. COCIVERA, F. C. KOKESH, V. MALATESTA and J. J. ZINCK, Catalysis of keto-enol tautomerism of oxaloacetic acid and its ions studied by proton nuclear magnetic resonance, J. Org. Chem. 12, 4076-4080 (1977).
280 18. 19. 20. 21. 22.
A. D. VINOGRADOV, et al. R . G . ANNET and G. W. KOSICKI, Oxaloacetate keto-enol tautomerase. Purification and characterization, J. Biol. Chem. 244, 2059-2067 (1969). J. C. WESENBERG, A. C H A U D H A R I and R. G. ANNET, Localization of oxaloacetate ketty-enol tautomerase, Can. J. Biochem. 54, 233-237 (1976). L. RYDEN, L.-C. OFVERSTEDT, H. BEINERT, M. H. E M P T A G E and M. C. KENNEDY, Molecular weight of beef heart aconitase and stoichiometry of the components of its iron-sulfur cluster, J. Biol. Chem. 259, 3141-3144 (1984). J . P . GLUSKER, Aconitase, pp. 413-439 in The Enzymes, Vol 5 (P. D. BOYER, ed.), Academic Press, Inc., New York (1971). D . J . KUO and I. A. ROSE, Aconitase: its source of catalytic protons, Biochemistry 26, 7589-7596 (1987).