Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.     

Crystalline NAD/NADP-dependent malate dehydrogenase; the enzyme from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

Malate dehydrogenase from Sulfolobus acidocaldarius has been purified 240-fold to apparent electrophoretic homogeneity. The enzyme shows a specific activity of 277 U/mg and crystallizes readily. The relative molecular mass of the native enzyme is estimated as 128,500 by ultracentrifugation. After cross-linking a relative molecular mass of 134,000 is found by sodium dodecyl sulfate gel electrophoresis. Malate dehydrogenase from S. acidocaldarius is composed of four subunits of identical size with a relative molecular mass of 34,000. Active-enzyme sedimentation in the analytical ultracentrifuge indicates that the tetramer is the catalytically active species. Kinetic studies in the direction of oxaloacetate reduction showed a Km for NADH of 4.1 microM and a Km for oxaloacetate of 52 microM. Oxaloacetate exhibits substrate inhibition at higher concentrations, L-malate, NAD and NADP were found to be product inhibitors. The enzymatic activity is inhibited by 2-oxoglutarate but not by the adenosine nucleotides AMP, ADP and ATP. Only low activity is detected in the direction of malate oxidation. Malate dehydrogenase from S. acidocaldarius utilizes both NADH and NADPH to reduce oxaloacetate. The enzyme shows A-side stereospecificity for both nicotinamide dinucleotides.     

Properties and function of malate enzyme from Pseudomonas putida

Malate enzyme (L-malate: NADP+ oxidoreductase (oxalacetate-decarboxylating, EC 1.1.1.40)) has been purified from Pseudomonas putida to 99 per cent homogeneity by heat, ammonium suphate fractionation, gel filtration and anion exchange chromatography. Sodium dodecylsulphate-(SDS)-polyacrylamide disc gel electrophoresis analysis showed an approximate tetrameric subunit with a molecular weight of 52,000. The purified enzyme showed a pH optimum between 8.0 and 8.5 (for Tris-HCl buffer) and required bivalent cations for catalysis; monovalent ions like K+ and NH4+ acted as very effective activators. The temperature-activity relationship for the malate enzyme from 35-80 degrees C showed broken Arrhenius plots with an inflexion at 65 degrees C. The enzyme halflife was 30s at 85 degrees C. The enzyme showed hyperbolic kinetics for both substrates with apparent Km values of 4.0 X 10(-4) M and 2.3 X 10(-5) M for L-malate and NADP+ respectively. From the study of the effects of some compounds on the enzyme, the physiological significance of those produced by fumarate, succinate and oxalacetate can be emphasized.     

Purification and properties of L-aspartate aminotransferase of Chlamydomonas reinhardtii

An enzyme which catalyzes the transamination of L-aspartate with 2-oxoglutarate has been purified 400-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. An apparent relative molecular mass of 138,000 was estimated by gel filtration. The enzyme is a dimer consisting of two identical subunits of Mr 65,000 each as deduced from PAGE/SDS studies. A stoichiometry of two molecules pyridoxal 5-phosphate/enzyme molecule was calculated. The enzyme has an isoelectric point of 8.48 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-aspartate. L-Aspartate or pyridoxal 5-phosphate, but not 2-oxoglutarate, protected the enzyme from heat inactivation. The purified enzyme was able to transaminate, although to a low extent, L-phenylalanine and L-tyrosine with 2-oxoglutarate, and L-serine, L-alanine and L-glutamine with oxaloacetate. L-Aspartate aminotransferase exhibited hyperbolic kinetics for 2-oxoglutarate and oxaloacetate, and nonhyperbolic behaviour for L-aspartate and L-glutamate. Apparent Km values were 0.55 mM for 2-oxoglutarate, 0.044 mM for oxaloacetate, 2.53 mM for L-aspartate and 3.88 mM for L-glutamate. Transamination of L-aspartate in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

The first archaeal L-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus: gene cloning and enzymological characterization

A gene encoding an L-aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus. After expression in Escherichia coli, the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about 48 kDa. Characterization revealed the enzyme to be a highly thermostable L-aspartate dehydrogenase, showing little loss of activity following incubation for 1 h at up to 80 degrees C. The optimum temperature for L-aspartate dehydrogenation was about 80 degrees C. The enzyme specifically utilized L-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor. The Km values for L-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor. The Km values for NAD and NADP were 0.11 and 0.32 mM, respectively. For reductive amination, the Km values for oxaloacetate, NADH and ammonia were 1.2, 0.014 and 167 mM, respectively. The enzyme showed pro-R (A-type) stereospecificity for hydrogen transfer from the C4 position of the nicotinamide moiety of NADH. This is the first report of an archaeal L-aspartate dehydrogenase. Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species.     

Purification and properties of mitochondrial malate dehydrogenase of Toxocara canis muscle

Mitochondrial malate dehydrogenase was purified from muscle extracts of Toxocara canis by means of Sephadex G-100 gel filtration, DEAE-Sephadex ion-exchange chromatography and 5'AMP-Sepharose 4B affinity chromatography. The purified enzyme showed an optimum pH for the reduction of oxaloacetate of 7.3 in Tris-HCl buffer and of pH 7.5-7.8 in phosphate buffer. The m-MDH showed values of 3.2 kcal/mol and 10.5 kcal/mol for the energy of activation, calculated from the Arrhenius equation. The mitochondrial enzyme was found to be more susceptible to thermal inactivation as compared with the cytosolic isoenzyme. Kinetic experiments showed that the m-MDH of Toxocara canis is inhibited by excess oxaloacetate but not by excess NADH. The apparent Km for oxaloacetate reduction was 53 microM and 0.54 mM for L-malate oxidation.     

Structural specificity of the allosteric inhibitor of phosphoenolpyruvate carboxylase of Escherichia coli.

The structural specificity of the allosteric inhibitor of phosphoenolpyruvate carboxylas [EC 4.1.1.31] of Escherichia coli W was investigated using native enzyme and photooxidized enzyme which was desensitized to L-aspartate. Inhibitory activity was expressed in terms of the concentration of the compound required for 50% inhibition (I0.5). For the native enzyme, L-aspartate and L-malate were the strongest inhibitors with I0.5 values of about 0.10-0.15 mM among about 20 componds tested. For the photooxidized enzyme, oxaloacetate and L-malate were relatively strong inhibitors wiht I0.5 values of about 11-16 mM. The results obtained suggest that the inhibition of the native enzyme mainly reflects allosteric inhibition.     

The identification and characterization of two cyclic nucleotide phosphodiesterases from bovine adrenal medulla

Two soluble cyclic nucleotide phosphodiesterase activities, designated Peak I (Mr = 216,000) and Peak II (Mr = 230,000), have been isolated from bovine adrenal medulla by DEAE-cellulose chromatography. Peak I has Ca2+-independent, cGMP-specific phosphodiesterase activity and Peak II has cGMP-stimulated cyclic nucleotide phosphodiesterase activity. Peak I hydrolyzes cGMP with hyperbolic kinetics and demonstrates a Km of 23 microM. Peak II hydrolyzes cGMP with hyperbolic kinetics but hydrolyzes cAMP with slightly sigmoidal kinetics and demonstrates Km values of 54 +/- 0.7 microM cGMP and 38 +/- 6 microM cAMP. Cyclic AMP and cGMP are competitive inhibitors of each other's hydrolysis, suggesting that these nucleotides may be hydrolyzed at the same catalytic site. Micromolar concentrations of cGMP cause a 5-fold stimulation of the hydrolysis of subsaturating concentrations of cAMP by the Peak II phosphodiesterase. Half-maximal activation occurs at 0.5 microM cGMP and the result of activation is a decrease in the apparent Km for cAMP. Stimulation of the hydrolysis of subsaturating concentrations of cGMP by cAMP was also detected; however, cAMP is a less potent activator of the enzyme than cGMP. Cyclic AMP causes a 1.5-fold stimulation of cGMP hydrolysis and half-maximal activation occurs at 2.5 microM cAMP.     

High activity of NADP-dependent malic enzyme in mitochondria from abdomen muscle of the crayfish Orconectes limosus.

1. Mitochondria isolated from abdomen muscle of crayfish Orconectes limosus exhibit malic enzyme activity in the presence of L-malate, NADP and Mn2+ ions after addition of Triton X-100. Under optimal conditions about 230 nmole of reduced NADP and an equivalent amount of pyruvate are produced per min per mg of mitochondrial protein. 2. The pH optimum for decarboxylation of L-malate is about 7.5. 3. The apparent Km for L-malate, NADP and Mn2+ ions was found to be 0.66, 0.012, and 0.0025 mM, respectively. 4. The requirement for Mn2+ can be replaced by Mg2+, Co2+ and Ni2+ ions; however, higher concentrations of these ions than Mn2+ are required for a full stimulation of malic enzyme activity. 5. Oxaloacetate and pyruvate inhibited the enzyme activity in a competitive manner with apparent Ki values of 0.05 mM and 5.4 mM, respectively.     

Human blood platelet 3': 5'-cyclic nucleotide phosphodiesterase. Isolation of low-Km and high-Km phosphodiesterase.

Human blood platelet contained at least three kinetically distinct forms of 3': 5'-cyclic nucleotide phosphodiesterase (3': 5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) (F I, F II, and F III) which were clearly separated by DEAE-cellulose column chromatography. Although a few properties of the platelet phosphodiesterases such as their substrate affinities and DEAE-cellulose profile resembled somewhat those of the three 3': 5'-cyclic nucleotide phosphodiesterase in rat liver reported by Russell et al. [10], there were pronounced differences in some properties between the platelet and the liver enzymes: (1) the platelet enzymes hydrolyzed both cyclic nucleotides and lacked a highly specific cyclic guanosine 3': 5'-monophosphate (cyclic GMP) phosphodiesterase and (2) kinetic data of the platelet enzymes indicated that cyclic adenosine 3': 5'-monophosphate (cyclic AMP) and cyclic GMP interact with a single catalytic site on the enzyme. F I was a cyclic nucleotide phosphodiesterase with a high Km for cyclic AMP and a negatively cooperative low Km for cyclic GMP. F II hydrolyzed cyclic AMP and cyclic GMP about equally with a high Km for both substrates. F III was low Km phosphodiesterase which hydrolyzed cyclic AMP faster than cyclic GMP. Each cyclic nucleotide acted as a competitive inhibitor of the hydrolysis of the other nucleotide by these three fractions with Ki values similar to the Km values for each nucleotide suggesting that the hydrolysis of both cyclic AMP and cyclic GMP was catalyzed by a single catalytic site on the enzyme. However, cyclic GMP at low concentration (below 10 muM) was an activator of cyclic AMP hydrolysis by F I. Papaverine and EG 626 acted as competitive inhibitors of each fraction with virtually the same Ki value in both assays using either cyclic AMP or cyclic GMP as the substrate. The ratio of cyclic AMP hydrolysis to cyclic GMP hydrolysis by each fraction did not vary significantly after freezing/thawing or heat treatment. These facts also suggest that both nucleotides were hydrolyzed by the same catalytic site on the enzyme. The differences in apparent Ki values for inhibitors such as cyclic nucleotides, papaverine and EG 626 would indicate that three enzymes were different from each other. Centrifugation in a continuous sucrose gradient revealed sedimentation coefficients F I and II had 8.9 S and F III 4.6 S. The molecular weight of these forms, determined by gel filtration on a Sepharose 6B column, were approx. 240 000 (F I and II) and 180 000 (F III). F III was purified extensively (70-fold) from homogenate, with a recovery of approximately 7%.     

Properties and function of malate enzyme from Dicentrarchus labrax L. liver

Malate enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate decarboxylating, EC 1.1.1.40) has been purified from Dicentrarchus labrax liver to 99% homogeneity by gel filtration, anion exchange and affinity chromatographies. The apparent molecular weight was estimated by gel filtration chromatography to be 148,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide disc gel electrophoresis was shown to be a tetrameric protein. The purified enzyme showed a pH optimum 8.5 (Tris-HCl buffer) and required bivalent cations for catalysis. The temperature-activity relationship for the enzyme showed broken Arrhenius plots with inflexions at 15 and 40 degrees C. Kinetic properties and the effects of some metabolites related to L-malate are studied.     

Properties and function of mitochondrial malate enzyme from bass liver

Malate enzyme (L-malate: NADP+ oxidoreductase oxaloacetate decarboxylating, EC 1.1.1.40) from bass liver mitochondria was purified to over 90% of homogeneity by gel filtration, affinity and ion exchange chromatographies. The apparent molecular weight estimated by gel filtration was 316,000. Analysis of the enzyme on sodium dodecylsulphate-polyacrylamide disc gel electrophoresis was shown to be a tetramere protein. The enzyme required bivalent cations for catalysis, (Mn2+ or Mg2+) and displayed a narrow pH optimum (8.4-8.6 for Tris-HCl buffer) and was inactivated by p-chloromercuribenzoate. The double reciprocal initial velocity plots of both of the substrates, NADP and malate, were linear and intercepting at a point that suggests a sequential mechanism. Product inhibition studies with NADP and malate as variable substrate are consistent with an ordered Bi-Ter mechanism.     

Purification and properties of glutamate synthase from Bacillus licheniformis

Glutamate synthase [L-glutamate:NADP+ oxidoreductase (transaminating); EC 1.4.1.13](GltS) was purified to homogeneity from Bacillus licheniformis A5. The native enzyme had a molecular weight of approximately 220,000 and was composed of two nonidentical subunits (molecular weights, approximately 158,000 and approximately 54,000). The enzyme was found to contain 8.1 +/- 1 iron atoms and 8.1 +/- 1 acid-labile sulfur atoms per 220,000-dalton dimer. Two flavin moieties were found per 220,000-dalton dimer, with a ratio of flavin adenine dinucleotide to flavin mononucleotide of 1.2. The UV-visible spectrum of the enzyme exhibited maxima at 263,380 and 450 nm. The GltS from B. licheniformis had a requirement for NADPH, alpha-ketoglutarate, and glutamine. Classical hyperbolic kinetics were seen for NADPH affinity, which resulted in an apparent Km value of 13 microM. Nonhyperbolic kinetics were obtained for alpha-ketoglutarate and glutamine affinities, and the reciprocal plots obtained for these substrates were biphasic. The apparent Km values obtained for glutamine were 8 and 100 microM, and the apparent Km values obtained for alpha-ketoglutarate were 6 and 50 microM. GltS activity was found to be relatively insensitive to inhibition by amino acids, keto acids, or various nucleotides. L-Methionine-DL-sulfoximine, L-methionine sulfone, and DL-methionine sulfoxide were found to be potent inhibitors of GltS activity, yielding I0.5 values of 150, 11, and 250 microM, respectively. GltSs were purified from cells grown in the presence of ammonia and nitrate as sole nitrogen sources and were compared. Both yielded identical final specific activities and identical physical (UV-visible spectra, flavin, and iron-sulfur composition) and kinetic characteristics.     

Malate oxidation by mitochondrial succinate:ubiquinone-reductase

Succinate:ubiquinone reductase was shown to catalyze the oxidation of L- and D-stereoisomers of malate by artificial electron acceptors and ubiquinone. The rate of malate oxidation by succinate:ubiquinone reductase is by two orders of magnitude lower than that for the natural substrate--succinate. The values of kinetic constants for the oxidation of D- and L-stereoisomers of malate are equal to: V infinity = 0.1 mumol/min/mg protein, Km = 2 mM and V infinity = 0.05 mumol/min/mg protein, Km = 2 mM, respectively. The malate dehydrogenase activity is fully inhibited by the inhibitors of the dicarboxylate-binding site of the enzyme, i.e., N-ethylmaleimide and malonate and is practically insensitive to carboxin, a specific inhibitor of the ubiquinone-binding center. The enol form of oxaloacetate was shown to be the product of malate oxidation by succinate:ubiquinone reductase. The kinetics of inhibition of the enzyme activity by the ketone and enol forms of oxaloacetate was studied. Both forms of oxaloacetate effectively inhibit the succinate:ubiquinone reductase reaction.     

Enzymatic properties of pyruvate kinase from the rumen ciliates genus Entodinium

1. Pyruvate kinase from the rumen ciliates genus Entodinium was partially purified and the enzymatic properties were investigated. 2. Three types of pyruvate kinase (type I, II and III) on DEAE-cellulose column were eluted with a linear gradient of KCl. The enzymatic properties differed among the types of enzyme, especially type I and type III displayed different kinetic properties to each other. The enzymatic property of type II enzyme was an intermediate between type I and III. 3. The principal enzyme, type I, required a divalent cation, Mg2+ and was activated with AMP and FDP. ATP was a potent inhibitor. The saturation curves for the substrates, PEP and ADP, were hyperbolic and Km values were 0.15 and 0.27 mM, respectively.     

Purification by affinity chromatography of a membrane dicarboxylate binding protein from Bacillus subtilis

Membrane vesicles of Bacillus subtilis W23 actively transport the C4 and C5 dicarboxylates of the tricarboxylate cycle by system(s) of relatively high affinity for their requisite substrates (Km 4-53 microM). Glutamate and succinate binding activities were readily solubilized from membrane vesicles by nonionic detergents, particularly by Lubrol WX. From this extract, glutamate binding activity was highly enriched by affinity chromatography on phloroglucinol-expanded Sepharose-6B to which L-aspartate was coupled via divinylsulfone. Another protein (41000 molecular weight), which bound both L-glutamate and L-malate, was purified from affinity columns to which either L-glutamate or L-malate had been coupled via bisdiglycidyl ether. This protein bound labelled L-malate as well as L-glutamate with affinities similar to those seen with membrane vesicles (Kd's 8 microM L-malate and 52 microM L-glutamate).     

The identification of a new cyclic nucleotide phosphodiesterase activity in human and guinea-pig cardiac ventricle. Implications for the mechanism of action of selective phosphodiesterase inhibitors.

Four cyclic nucleotide phosphodiesterase (PDE) activities were separated from low-speed supernatants of homogenates of human cardiac ventricle by DEAE-Sepharose chromatography, and designated PDE I-PDE IV in order of elution with an increasing salt gradient. PDE I was a Ca2+/calmodulin-stimulated activity, and PDE II was an activity with a high Km for cyclic AMP which was stimulated by low concentrations of cyclic GMP. Human ventricle PDE III had Km values of 0.14 microM (cyclic AMP) and 4 microM (cyclic GMP), and showed simple Michaelis-Menten kinetics with both substrates. PDE IV is a previously unrecognized activity in cardiac muscle, the human enzyme having Km values of 2 microM (cyclic AMP) and 50 microM (cyclic GMP). PDE III and PDE IV were not activated by cyclic nucleotides or calmodulin. Four PDE activities were also isolated from guinea-pig ventricle, and had very similar kinetic properties. By gel filtration, the Mr of PDE III was 60,000, and that of PDE IV 45,000. The drug SK&F 94120 selectively and competitively inhibited PDE III with a Ki value of 0.8 microM (human), showing simple hyperbolic inhibition kinetics. Rolipram (Schering ZK 62711) and Ro 20-1724 (Roche), which have previously been reported to inhibit PDE III-like activities strongly, were shown to be weak inhibitors of human and guinea-pig PDE III enzymes (Ki values greater than 25 microM), but potent inhibitors of PDE IV [Ki values 2.4 microM (Rolipram) and 3.1 microM (Ro 20-1724) with human PDE IV]. The inhibition in all cases demonstrated simple hyperbolic competition. These observations suggest that the previously reported complex inhibition of PDE III-type activities from cardiac muscle was caused by incomplete separation of the PDE III from other enzymes, particularly PDE IV.     

Regulation of biosynthesis of secondary metabolites

The process of isolation and purification of malate dehydrogenase (decarboxylating) (EC 1.1.1.40) from the mycelium of the actinomycete Streptomyces aureofaciens has been worked out. The enzyme was purified 35 fold. The kinetic characters of the purified enzyme are very similar to the figures for malate dehydrogenase (decarboxylating) from other sources. Km for L-malate = 2.1 X 10(-3)M, Km for NADP = 4.6 X 10(-5)M (at pH 7.4). The reaction requires metal divalent ions, Mn2+ being more effective than Mg2+. The enzyme reaches its maximal activity at pH 8.75.     

Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.     

Effect of photooxidation on catalytic and regulatory properties of NAD-linked malic enzyme from Escherichia coli

In an aim to elucidate the structure-function relationship of NAD-linked malic enzyme [EC 1.1.1.38] from Escherichia coli W, the effect of chemical modification on the catalytic and regulatory properties of the enzyme was studied. Upon photooxidation of the enzyme in the presence of methylene blue, a time-dependent inactivation occurred following pseudo-first order kinetics. The pH-dependence of the inactivation rate exhibited a pK value of 6.1. L-Malate, NAD+, and Mn2+ markedly protected the enzyme against the inactivation. Prior masking of the catalytically essential sulfhydryl groups with p-mercuribenzoate did not result in a retardation of the rate of photoinactivation. This excluded the possibility of an involvement of sulfhydryl group modification in the photoinactivation. Although the Km values for L-malate and NAD+ were not affected by photooxidation, the S0.5 value and the Hill coefficient for Mn2+ were considerably altered, and the cooperative nature of the saturation profile for Mn2+ in the native enzyme was completely abolished. The activating effect of L-aspartate on the native enzyme was completely abolished upon photooxidation, and the inhibitory effect of CoA was also diminished to a marked extent upon the treatment. The oxaloacetate decarboxylating activity of the enzyme was lost in parallel with the loss of the activity for oxidative decarboxylation of L-malate. These results suggest a possible involvement of histidyl residue(s) in the catalytic and regulatory functions of the enzyme.