Spectroscopic investigation of binary and ternary coenzyme complexes of yeast alcohol dehydrogenase.

Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.     

Half-site reactivity of an essential thiol group of D-beta-hydroxybutyrate dehydrogenase

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme, which is a tetramer both in the mitochondrial inner membrane and as the purified enzyme reconstituted with phospholipid. For the active enzyme-phospholipid complex in the absence of ligands, we previously found that reaction with N-ethylmaleimide (at 5 mol/mol of enzyme subunit) resulted in progressive loss of enzymic activity with an inactivation stoichiometry of 1 equiv of sulfhydryl derivatized per mole of enzyme and a maximum derivatization of 2 equiv [Latruffe, N., Brenner, S. C., & Fleischer, S. (1980) Biochemistry 19, 5285-5290]. We now find, in the presence of nucleotide or substrate, that the rate of inactivation is significantly reduced, which indicates that these ligands afford protection of the essential sulfhydryl. Further, in the presence of ligands, the inactivation stoichiometry is 0.5, consistent with half-of-the-site reactivity of the essential sulfhydryl. Thus, at a low ratio of N-ethylmaleimide to enzyme, nucleotide or substrate affords essentially complete protection of the nonessential sulfhydryl from derivatization. The binding characteristics of NADH to both the native and N-ethylmaleimide-derivatized enzyme have been compared by fluorescence spectroscopy. Quenching of intrinsic tryptophan fluorescence of the protein shows that the enzyme, derivatized with N-ethylmaleimide either in the absence or in the presence of NAD+, binds NADH but with a reduced Kd (approximately 50 microM as compared with approximately 20 microM for native enzyme). However, a critical change has occurred in that resonance energy transfer from protein to bound NADH, observed in the native enzyme, is abolished in the N-ethylmaleimide-derivatized enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical evidence for the reversed polarity of the outer membrane of the bacterial forespore.

Measurement of certain membrane-bound enzymic activities was used to study the orientation of the outer membrane of the double-membraned forespore of Bacillus megaterium KM. 2. Adenosine triphosphatase, NADH dehydrogenase and L-malate intact protoplasts, but were readily detected in intact stage II or IV forespores, consistent with reversed polarity of the outer forespore membrane relative to the mother-cell plasma membrane. 3. Measurement of NADH oxidase activity revealed that intact stage III forespores had the same high affinity for NADH as protoplast membrane preparations and protoplast lystates, consistent with ready access of NADH to oxidation sites on the outer forespores membrane. 4. Forespores and protoplasts showed osmometric behaviour in solutions of non-permanent solutes consistent with the presence of an intact permeability barrier in these structures.     

The first examples of (S)-2-hydroxyacid dehydrogenases catalyzing the transfer of the pro-4S hydrogen of NADH are found in the archaea.

Reduction of 2-oxoacids to the corresponding (S)-2-hydroxyacids is an important transformation in biochemistry. To date all (S)-2-hydroxyacid dehydrogenases belonging to the L-lactate/L-malate dehydrogenase family have been found to transfer the pro-4R hydrogen of either NADH or NADPH to C-2 of the 2-oxoacid substrates during their reduction. Here, we report that recombinantly generated (S)-2-hydroxyacid dehydrogenases present in the methanoarchaea Methanococcus jannaschii and Methanothermus fervidus use the pro-4S hydrogen of NADH to reduce a series of 2-oxoacids to the corresponding (S)-2-hydroxyacids. This information as well as the low sequence identity between these archaeal enzymes and the L-lactate/L-malate family of enzymes indicate that these enzymes are not evolutionary related and therefore constitute a new class of (S)-2-hydroxyacid dehydrogenases.     

Mitochondrial NADH fluorescence is enhanced by complex I binding

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several-fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10-fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state.     

Biochemical studies of supernatant malate dehydrogenase allozymes in Drosophila melanogaster.

1. A biochemical comparison was made among cytoplasmic malate dehydrogenase allozymic variants from Drosophila melanogaster. Experiments were carried out on enzyme extracted from six different genotypes: three homozygotes and their respective heterozygotes. 2. The allozyme forms (MDH A, MDH B, MDH C) were indistinguishable in terms of NAD and L-malate optima, while they are distinguishable in terms of NADH and OAA saturation conditions. Activities were inhibited at concentrations greater than 0.36 and 0.40 mM NADH for BB and AA, CC, respectively, while in relation to OAA inhibition was observed at concentrations higher than 3 or 6 mM for the AA, CC and BB, respectively. 3. differences among genotypes were also observed in thermal stability: Km values for OAA, L-malate, NADH and NAD: and pG optima. 4. A simple method is presented for the separation of the cytoplasmic from the mitochondrial malate dehydrogenase.     

Studies on the mechanism of the malate dehydrogenase reaction.

The stereospecificity of the chicken heart mitochondrial malate dehydrogenase as well as the ability of this enzyme to form various abortive complexes has been further investigated. The enzyme was found to be specific for the A-hydrogen of NADH. Complex formation of the enzyme with oxalacetate and oxidized coenzymes is pH-dependent and is promoted at alkaline pH values. The enol form of oxalacetate appears to be the species that participates in the formation of the complexes. The binding of L-malate, D-malate, or hydroxymalonate to the enzyme. NADH complex is also pH-dependent, and involves a group on the enzyme with a pK of 7.5. The binding of L-malate is promoted at alkaline pH values, whereas the binding of D-malate and hydroxymalonate is favored at acidic pH values. These results indicate that L-malate and enol-oxalacetate preferentially or exclusively bind to the nonprotonated form of the enzyme, whereas keto-oxalactate, hydroxymalonate, and D-malate only bind to the protonated form of the enzyme. Based on this conclusion, a detailed chemical mechanism for the malate dehydrogenase reaction has been postulated and a schematic illustration of the transition state of the enzyme is presented.     

The purification and steady-state kinetic behaviour of rabbit heart mitochondrial NAD(P)+ malic enzyme.

The mitochondrial NAD(P)+ malic enzyme [EC 1.1.1.39, L-malate:NAD+ oxidoreductase (decarboxylating)] was purified from rabbit heart to a specific activity of 7 units (mumol/min)/mg at 23 degrees C. A study of the reductive carboxylation reaction indicates that this enzymic reaction is reversible. The rate of the reductive carboxylation reaction appears to be completely inhibited at an NADH concentration of 0.92 mM. A substrate saturation curve of this reaction with NADH as the varied substrate describes this inhibition. The apparent kinetic parameters for this reaction are Ka(NADH) = 239 microM and Vr = 1.1 mumol/min per mg at 23 degrees C. The steady-state product-inhibition patterns for pyruvate and NADH indicate a sequential binding of the substrates: NAD+ followed by L-malate. These data also indicate that NADH is the last product released. A steady-state kinetic model is proposed that incorporates NADH-enzyme dead-end complexes.     

Studies of enzyme-ligand complexes using dynamic fluorescence anisotropy. II. The coenzyme-binding site of malate dehydrogenase

The coenzyme-binding site in mitochondrial malate dehydrogenase from pig heart was studied using dynamic fluorescence anisotropy decay. The dynamics of the fluorescent ligands NADH and 6-cyano-7-hydroxy-4,8-dimethylcoumarin were used to detect conformational changes at the dihydronicotinamide-and at the adenosine-binding sites, respectively. Addition of the natural substrate L-malate to the complex from enzyme and NADH does not influence the complete immobilization of the dihydronicotinamide group, whereas the stereoisomer D-malate and the substrate-analogue hydroxymalonate form ternary complexes with highly mobile dihydronicotinamide. The dynamics of the fluorescent adenosine-analogue are not influenced by formation of complexes with substrate and substrate-analogues. Thus the conformational changes at the dihydronicotinamide-binding site remain local and are not transmitted to the adenosine-binding site.     

Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase.

1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows 'all-of-the-sites' behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate dehydrogenase and this, and the structural results of others, can be explained if the two enzymes are a product of divergent evolution.     

Malic enzyme activity and cyanide-insensitive electron transport in plant mitochondria.

In the presence of exogenous NAD⁺, malate oxidation by cauliflower mitochondria takes place essentially via an electron transport pathway that is insensitive to rotenone, antimycin and cyanide but is strongly sensitive to salicyl hydroxamic acid. It bypasses all phosphorylation sites. NAD⁺ is reduced by an enzyme identified as malic enzyme (L-malate:NAD oxidoreductase (decarboxylating), EC 1.1.1.39). The NADH produced is reoxidized by an internal rotenone-insensitive NADH dehydrogenase that yields electrons directly to the cyanide-insensitive pathway.     

Energization of mitochondrial inner membranes caused by l-malate

It was found that 0.06 μg antimycin A/mg mitochondrial protein, an amount sufficient to inhibit electron transfer between cytochromes b and c₁ completely, fully reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. The effect of L-malate on cytochrome a was insensitive to oligomycin, but all the uncouplers and detergents tested reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. It was also found that addition of L-malate to anaerobic mitochondria, like addition of ATP, decreased the fluorescence of 1-anilinonaphthalene-8-sulphonate, and that subsequent addition of uncouplers reversed this effect. The effect of L-malate on the fluorescence of the dye was insensitive to oligomycin. The present findings suggest that addition of L-malate may cause energization of the mitochondrial inner membranes and that the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria may result from an L-malate-induced, energy-linked reversal of electron transfer in site II.     

Malate dehydrogenase. Kinetic studies of substrate activation of supernatant enzyme by L-malate.

At pH 8.0 in 0.05 M Tris-acetate buffer at 25 degrees C, homogeneous supernatant malate dehydrogenase exhibits substrate activation by L-malate. The turnover number, Michaelis constant for L-malate, and Michaelis constant for NAD are: 0.46 X 10(4) min(-1), 0.036 mM, and 0.14 mM, respectively, for nonactivated enzyme and 1.1 X 10(4) min(-1), 0.2mM, and 0.047 mM for the same series of constants in activated enzyme. Nonactivating behavior is observed at concentrations between 0.02 and 0.15 mM L-malate and activating behavior is observed between 0.15 and 0.5 mM L-malate. L-Malate activation is compared with similar activation of mitochondrial malate dehydrogenase. While it is not possible to exclude unequivocally all mechanisms, the data seem to be consistent with the occurrence of a fundamentally ordered bi bi mechanism, possibly involving activation through the allosteric binding of L-malate. It is concluded that the data are consistent with a form of the "reciprocating compulsory order mechanism" in which nonactivated enzyme reflects catalysis by one subunit and activated catalysis expresses the coordinated activity of two subunits. The allosteric interaction and the "reciprocating mechanism/ are not mutually exclusive.     

Subunit interactions in rabbit-muscle glyceraldehyde-phosphate dehydrogenase, as measured by NAD+ and NADH binding.

1. The binding parameters for NADH and NAD+ to rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been measured by quenching of the flourescence of the protein and the NADH. 2. The fact that the degree of protein fluorescence quenching by bound NAD+ or NADH, excited at 285 nm and measured at 340 nm ('blue' tryptophans), is not linearly related to the saturation functions of these nucleotides, leads to a slight overestimation of the interaction energy and an underestimation of the concentration of sites, if linearity is assumed. 3. This is also the case for NADH, but not for NAD+, when the protein fluorescence is excited at 305 nm and measured at 390 nm ('red' tryptophans). 4. The binding of NAD+ can be described by a model in which the binding of NAD+, via negative interactions within the dimer, induces weaker binding sites, with the result that the microscopic dissociation constant is 0.08 microM at low saturation and 0.18 microM for the holoenzyme. 5. The binding of NADH can be described on the basis of the same model, the dissociation constant at low saturation being 0.5 microM and of the holoenzyme 1.0 microM. 6. The fluorescence of bound NADH is not sensitive to the conformational changes that cause the decrease in affinity of bound NAD+ or NADH. 7. The binding of NAD+ to the 3-phosphoglyceroyl enzyme can be described by a dissociation constant that is at least two orders of magnitude greater than the dissociation constants of the unacylated enzyme. The affinity of NAD+ to this form of the enzyme is in agreement with the Ki calculated from product inhibition by NAD+ of the reductive dephosphorylation of 1,3-diphosphoglycerate.     

Action of surfactants on porcine heart malate dehydrogenase isoenzymes and a simple method for the differential assay of these isoenzymes

The cationic surfactant, cetyl (hexadecyl) trimethylammonium bromide (CTAB), completely inactivates porcine heart cytoplasmic malate dehydrogenase (L-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) at concentrations (of surfactant) which do not affect the activity of the mitochondrial isoenzyme. These concentrations are close to, or higher than, the critical micelle concentration of CTAB. An increase in the ionic strength of the medium significantly retards the CTAB-induced inactivation of the cytoplasmic enzyme. The enzyme is also markedly protected against CTAB inactivation by NADH; L-malate on its own has no effect but a combination of NADH and L-malate affords greater protection than NADH alone. The CTAB inactivation is not reversed by dilution of the surfactant. The highly selective action of CTAB on the two malate dehydrogenases, which correlates well with their electrostatic charges, has been exploited for a simple and reliable differential assay of these isoenzymes. The anionic surfactant, sodium dodecyl sulphate (SDS), at concentrations well below the critical micelle concentration, inactivates both isoenzymes, but the mitochondrial enzyme is significantly more sensitive than its cytoplasmic counterpart. There is thus some correlation, though not as strong as with CTAB, between SDS inactivation and the charges of the two malat dehydrogenases. An increase in ionic strength has opposite effects on the two isoenzymes: the mitochondrial enzyme becomes more resistant and the cytoplasmic enzyme less so. Both isoenzymes are rendered more resistant to SDS by the inclusion of NADH. Inactivation of the enzymes caused by short exposure to SDS is largely reversed by dilution of the detergent, but longer exposure leads to progressive irreversible loss of activity. NADH very effectively protects the isoenzymes against irreversible inactivation. It is likely that a reversible phase of inactivation precedes an irreversible phase and that in the former phase SDS acts competitively with NADH. Both malate dehydrogenases possess considerable resistance to the nonionic detergent, Triton X-100.     

Fluorescence investigations of coenzyme and substrate interactions in mannitol-1-phosphate dehydrogenase ofEscherichia coli

Coenzyme and substrate interactions with mannitol-1-phosphate dehydrogenase fromEscherichia coli (a dimer of MW 45,000) have been studied by fluorescence spectroscopy. NAD⁺ quenches the fluorescence emission of the protein tryptophan residues; shifting the excitation wavelength from 280 to 290 nm results in an increase in this quenching and a red shift in the emission maximum. NAD⁺ also quenches the fluorescence of covalently attached pyridoxyl phosphate, and this quenching is accompanied by a spectral broadening above 425 nm. Fructose-6-phosphate increases the binding of NAD⁺, but causes a slight reduction in the quenching of the tryptophan fluorescence observed at saturating levels of coenzyme, and reverses the NAD⁺-induced broadening in the pyridoxyl phosphate emission spectrum. NADH quenches the protein emission much less than NAD⁺; this quenching is not changed by shifting the excitation wavelength and is not affected by the presence of bound mannitol-1-phosphate. Titrations monitoring the quenching by NADH indicate a single class of NADH binding sites, while titrations monitoring NADH fluorescence suggest that coenzyme fluorescence is more enhanced when NADH is bound to less than half of the total enzyme subunits, with the emission per NADH molecule bound decreasing as the number of NADH molecules bound increases. In the absence of coenzyme, neither fructose-6-phosphate nor mannitol-1-phosphate have any effect on the protein tryptophan emission; however, both substrates induce specific changes in the emission spectrum of covalently attached pyridoxyl phosphate. These results suggest that the different coenzymes and substrates cause specific conformational changes in mannitol-1-phosphate dehydrogenase.     

Calcium transport in bovine sperm mitochondria: effect of substrates and phosphate.

Calcium uptake into filipin-treated bovine spermatozoa is completely inhibited by the uncoupler CCCP or by ruthenium red. Both Pi and mitochondrial substrates are required to obtain the maximal rate of calcium uptake into the sperm mitochondria. Bicarbonate and other anions such as lactate, acetate or beta-hydroxybutyrate do not support a high rate of calcium uptake. There are significant differences among various mitochondrial substrates in supporting calcium uptake. The best substrates are durohydroquinone, alpha-glycerophosphate and lactate. Pyruvate is a relatively poor substrate, and its rate can be greatly enhanced by malate or succinate but not by oxalacetate or lactate. This stimulation is blocked by the dicarboxylate translocase inhibitor, butylmalonate and can be mimiced by the non-metabolized substrate D-malate. The Ka for pyruvate was found to be 17 microM and 67 microM in the presence and absence of L-malate, respectively. The Ka for L-malate is 0.12 mM. It is suggested that in addition to the known pyruvate/lactate translocase there is a second translocase for pyruvate which is malate/succinate-dependent and does not transport lactate. In the presence of succinate, glutamate stimulates calcium uptake 3-fold, and this effect is not inhibited by rotenone. In the presence of glutamate plus malate or oxalacetate there is only an additive effect. It is suggested that glutamate stimulates succinate transport and/or oxidation in bovine sperm mitochondria. The alpha-hydroxybutyrate is almost as good as lactate in supporting calcium uptake. Since the alpha-keto product is not further metabolized in the citric acid cycle, it is suggested that lactate can supply the mitochondrial needs for NADH from its oxidation to pyruvate by the sperm lactate dehydrogenase x. Thus, when there is sufficient lactate in the sperm mitochondria, pyruvate need not be further metabolized in the citric acid cycle in order to supply more NADH.     

Inhibition of the malic enzyme from Acinetobacter calcoaceticus by NADPH and NADH]

The malic enzyme enriched from Acinetobacter calcoaceticus is inhibited by NADPH and NADH. The inhibition afforded by the reduced coenzymes is not affected by NAD+, AMP and 3'.5'-AMP. Against L-malate, NADPH inhibits the enzyme in a noncompetitive linear fashion (Ki = 1.5 x 10(-4) M), against NADP+, competitively linearly (Ki = 5.0 x 10(-5) M). While NADPH acted as a product inhibitor, NADH seems to be an allosteric effector of the malic enzyme, because with L-malate as the variable substrate in the double reciprocal plot, a nonlinear curve is obtained.     

The stereospecificity,purification, and characterization of an NADH-ferricyanide reductase from spinach leaf plasma membrane

Summary The stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane, peroxisomal membrane, plasma membrane and tonoplast are all specific for the -hydrogen of NADH whereas the reductases in the ER, the Golgi and the outer mitochondrial membrane are -specific. This shows unequivocally that the NADH-ferricyanide activity in the plasma membrane is not caused by ER contamination. In all the membranes one or several polypeptides with an apparent size of 45–50 kDa cross-react with antibodies raised against a microsomal NADH-ferricyanide reductase. An NADH-ferricyanide reductase was purified from spinach leaf plasma membranes. The enzyme was released from the membrane by CHAPS solubilization and purified 360-fold by ion-exchange chromatography followed by affinity chromatography and size exclusion chromatography on FPLC. A major band of 45 kDa was detected by SDS-PAGE and it cross-reacted with the anti-NADH-ferricyanide reductase antibodies. The native size of the enzyme is 160 kDa as determined by size-exclusion chromatography indicating that it is a tetramer. Isoelectric focusing revealed three isoenzymes between pH 5.3 and 5.6. The enzyme shows typical FAD fluorescence spectra with excitation peaks at 371 and 468 nm and an emission peak at 525 nm. It is specific for the -hydrogen of NADH and prefers NADH over NADPH as electron donor. It is highly specific for ferricyanide as electron acceptor and it is therefore unlikely to be the enzyme responsible for iron reduction on the outer surface of the plasma membrane.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate - DQ duroquinone - FPLC fast protein liquid chromatography; Ferricyanide hexacyanoferrate(III) - NEM N-ethylmaleimide - PCMB p-chloromercurobenzoate - SHAM salicylhydroxamic acid - SMP submitochondrial particles     

Selective chemical modification of arginine residues in mitochondrial malate dehydrogenase

Mitochondrial malate dehydrogenase (L-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) from porcine heart exhibits a time dependent loss in enzymatic activity in the presence of the reagent butanedione. The inhibition occurs concomitant with the modification of 2.4 residues of arginine per molecular weight of 70,000. The presence of the reduced coenzyme, NADH, protects the enzyme from inhibition by butanedione and from modification of arginine residues, suggesting that the residues modified are located near the coenzyme binding site and hence at or near the enzymatic active center of this enzyme.