Inhibition of glutamate dehydrogenase and malate dehydrogenases by palmitoyl coenzyme A.

In extension of a previous study with yeast glucose-6-P dehydrogenase (Kawaguchi, A., and Bloch, K. (1974) J. Biol. Chem. 249, 5793-5800), the structural changes accompanying the inhibition of glutamate dehydrogenase and several malate dehydrogenases by palmitoyl-CoA and by sodium dodecyl sulfate have been investigated. Palmitoyl-CoA converts liver glutamate dehydrogenase to enzymatically inactive dimeric subunits (Mr = 1.2 X 10(5)) and tightly binds to the dissociated enzyme. Removal of the inhibitor from the palmitoyl-CoA-dimer complex fails to regenerate enzyme activity. The Ki values for palmitoyl-CoA inhibition of malate dehydrogenases (oxalacetate reduction) are, for the enzyme from pig heart mitochondria, 1.8 muM, 500 muM from pig heart supernatant, and 10 muM from chicken heart supernatant. These inhibitions are readily reversible. Palmitoyl-CoA does not alter the quaternary structure of any of the malate dehydrogenases and binds only weakly to these enzymes. Mitochondrial malate dehydrogenase assayed in the direction malate to oxalacetate is much less sensitive to palmitoyl-CoA, with Ki values of 50 muM at pH 10 and greater than 50 muM at pH 7.4. While the differences in palmitoyl-CoA sensitivity in the forward and backward reactions catalyzed by mitochondrial dehydrogenase are unexplained, a physiological rationale for these differential effects is offered. Sodium dodecyl sulfate dissociates the various dehydrogenases to monomeric subunits in contrast to the more selective effects of palmitoyl-CoA.     

The nature of the substrate inhibition of cytoplasmic malate dehydrogenase from Phycomyces blakesleeanus

The mechanism that leads to substrate inhibition of cytoplasmic malate dehydrogenase (l-malate:NAD oxidoreductase, EC 1.1.1.37) from mycelium of Phycomyces blakesleeanus was investigated. A dead-end complex between enzyme and the enol-oxalacetate form appears to be responsible for this inhibition. The formation of the complex occurs more readily at acidic pH values. Results of this study suggest that the binding of oxalacetate as inhibitor prevent the NADH binding, with an estimated K_i for oxalacetate of 1.8 ± 0.1 mM. Such a competitive inhibition implies that the binding site for oxalacetate is the same both as inhibitor as well as substrate.     

A comparison of the malate dehydrogenase of the propionic acid bacteria with the mammalian soluble and mitochondrial isoenzymes.

1. Like the malate dehydrogenases of eucaryotic cells, the Propionibacterium shermanii enzyme is a dimer consisting of two 35,000 molecular weight subunits. 2. In electrophoretic behavior, resistance to substrate inhibition and stability to heating and dilution the P. shermanii MDH is more similar to the s-MDH than to the m-MDH of pig heart. 3. The P. shermanii MDH has a high turnover number (ca. 140,000) as well as Km values for both L-malate and oxalacetate which are four times higher than the mammalian isoenzymes. 4. A coupled assay for MDH using the malate-lactate transhydrogenase and diaphorase is described in which both substrates, L-malate and NAD, are regenerated.     

Substrate channeling of oxalacetate in solid-state complexes of malate dehydrogenase and citrate synthase

Current evidence suggests that mitochondrial matrix enzymes exist in solid-state, multienzyme complexes in vivo. Addition of polyethylene glycol to a solution containing malate dehydrogenase and citrate synthase generates such a solid-state, enzyme complex in vitro at enzyme concentrations permitting kinetic measurements. Suspensions of the isolated, solid-state, hetero-complex of these enzymes were used to study the coupled reactions of citrate synthesis from malate, NAD, and CoASAc. The particles appear to be about 1 microgram in diameter. Considering the ratio of enzyme to oxalacetate molecules in or at the surface of the solid-state particles, one would expect oxalacetate to be converted to citrate within a few molecular distances of the site of oxalacetate generation. This model of "substrate channeling" (or alternatively a direct transfer of oxalacetate between enzymes) is supported by experiments with excess aspartate aminotransferase and glutamate added to the solution phase to give a reaction competing with the synthase for bulk phase oxalacetate. Quantities of aminotransferase that reduce the citrate reaction rate with soluble dehydrogenase and synthase by 90% do not significantly affect rates with comparable amounts of the dehydrogenase-synthase complex. We suggest that similar substrate channeling can occur in vivo and discuss the possible advantages provided thereby.     

Kinetic studies on pig heart cytoplasmic malate dehydrogenase.

Kinetic studies on the pig heart cytoplasmic malate dehydrogenase have been performed over a wide range of conditions using the full time course of the reaction and computer simulation to obtain the kinetic parameters. The maximum velocity and Michaelis constants for the oxidation of reduced coenzyme have been determined as a fundtion of pH in 0.05 M phosphate buffer at 15 degrees. At pH 7.5 and at low substrate concentrations, the kinetic data are consistent with a sequential addition of substrates, coenzyme binding first, and involving the formation of at least one ternary complex. No oxalacetate binding to the enzyme was observed. The rate constants for the dissociation of coenzyme from the enzyme-coenzyme complex are small enough to define the maximum velocity in either direction of the reaction. These data, plus data using deuterated reduced coenzyme, indicate that the chemical transformation step is not rate determining. It is also shown that DPNH binding can be tight enough to practically exclude the possibility of obtaining initial velocities when measuring the reduction of DPN. Kinetic abnormalities do appear at higher substrate or product concentrations, but these do not appear to be related to the formation of inactive abortice, complexes.     

Resistance of tropoelastin and elastin peptides to degradation by alpha 2-macroglobulin-protease complexes

When α-ketoglutarate is the substrate, malate is a considerably more effective inhibitor of glutamate dehydrogenase than glutamate, oxalacetate, aspartate, or glutarate. Malate is a considerably poorer inhibitor when glutamate is the substrate. Malate is competitive with α-ketoglutarate, uncompetitive with TPNH, and noncompetitive with glutamate. The above, plus the fact that malate is a considerably more potent inhibitor when TPNH rather than TPN is the coenzyme, indicates that malate is predominantly bound to the α-ketoglutarate site of the enzyme-TPNH complex and has a considerably lower affinity for the enzyme-TPN complex. Ligands which decrease binding of TPNH to the enzyme such as ADP and leucine markedly decrease inhibition by malate. Conversely, GTP, which increases binding of TPNH to the enzyme also enhances inhibition by malate. Malate also decreases interaction between mitochondrial aspartate aminotransferase and glutamate dehydrogenase. This effect of malate on enzyme-enzyme interaction is enhanced by DPNH and GTP which also increase inhibition of glutamate dehydrogenase by malate and is decreased by TPN, ADP, ATP, α-ketoglutarate, and leucine which decrease inhibition of glutamate dehydrogenase by malate. These results indicate that malate could decrease α-ketoglutarate utilization by inhibiting glutamate dehydrogenase and retarding transfer of α-ketoglutarate from the aminotransferase to glutamate dehydrogenase. These effects of malate would be most pronounced when the mitochondrial level of α-ketoglutarate is low and the level of malate and reduced pyridine nucleotide is high.     

Chemical modification of the essential arginine in malate dehydrogenases

Chemical modification of arginine in malate dehydrogenases from pig heart mitochondria and from Bacillus subtilis was done using 4-hydroxy-3-nitrophenylglyoxal. Incorporation of 2 reagent molecules per subunit was observed concomitantly with complete loss of enzymatic activity. Partial protection was obtained with a substrate analogue and by formation of abortive ternary complexes, whereas coenzyme alone did not inhibit the inactivation. Modified inactive enzymes formed binary complexes with coenzyme as well as the ternary complex with NAD/sulfite. The substrate analogue 8-hydroxy-1,3,6-pyrenetrisulfonate was bound with reduced affinity, however. Because of the known stoichiometry of two reagent molecules per arginine we conclude that one arginine essential for substrate binding was modified in both enzymes.     

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.     

Nicotinamide 3,N4-ethenocytosine dinucleotide, an analog of nicotinamide adenine dinucleotide. Synthesis and enzyme studies.

A structural analog of NAD+, NICOTINAMIDE 3,N-4ethenocytosine dinucleotide (epsilonNCD+), has been synthesized, characterized, and compared in activity with the natural coenzyme in several enzyme systems. The Vmax and apparent Km values were determined for NAD+, epsilonNCD+, and epsilonNAD+ (nicotinamide 1, N6-ethenoadenine dinucleotide) with yeast alcohol, horse liver alcohol, pig heart malate, beef liver glutamate, and rabbit muscle lactate and glyceraldehyde-3-phosphate dehydrogenases. The Vmax for epsilonNCD+ was as great or greater than that obtained for NAD+ with three of the enzymes, 60-80 per cent with two others, and 14 percent with one. EpsilonNCD+ was found to be more active than epsilonNAD+ with all six dehydrogenases. EpsilonNCD+ served as a substrate for Neurospora crassa tnadase, but could not be phosphorylated with pigeon liver NAD+ kinase. NAD+ pyrophosphorylase from pig liver was unable to catalyze the formation of epsilonNCD+ from the triphosphate derivative of epsilon-cytidine and nicotinamide mononucleotide, but was able to slowly catalyze the pyrolytic cleavage of epsilonNCD+. The coenzyme activity of epsilonNCD+ with dehydrogenases can be discussed in terms of the close spatial homology of epsilonNCD+ and NAD+, which may allow similar accommodations within the enzyme binding regions.     

Purification, characterization, and overexpression of psychrophilic and thermolabile malate dehydrogenase of a novel antarctic psychrotolerant, Flavobacterium frigidimaris KUC-1

We purified the psychrophilic and thermolabile malate dehydrogenase to homogeneity from a novel psychrotolerant, Flavobacterium frigidimaris KUC-1, isolated from Antarctic seawater. The enzyme was a homotetramer with a molecular weight of about 123 k and that of the subunit was about 32 k. The enzyme required NAD(P)(+) as a coenzyme and catalyzed the oxidation of L-malate and the reduction of oxalacetate specifically. The reaction proceeded through an ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment, and the half-life time at 40 degrees C was estimated to be 3.0 min. The k(cat)/K(m) (microM(-1).s(-1)) values for L-malate and NAD(+) at 30 degrees C were 289 and 2,790, respectively. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of the coenzyme. The enzyme contained 311 amino acid residues and much lower numbers of proline and arginine residues than other malate dehydrogenases.     

Malate dehydrogenase: a model for structure, evolution, and catalysis.

Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.     

Histidine residues and the enzyme activity of pig heart supernatant malate dehydrogenase (Short Communication)

1. Supernatant pig heart malate dehydrogenase is completely inhibited by reaction with diethyl pyrocarbonate at pH6.5, when 0.58+/-0.1 residue of ethoxycarbonylhistidine is formed per NADH-binding site. 2. Oxaloacetate and hydroxymalonate protect the enzyme from inhibition in the absence of coenzyme. 3. Limited ethoxycarbonylation does not alter the binding of NADH to the enzyme but prevents the enzyme-NADH complex from interacting with hydroxymalonate in a ternary complex.     

Structural basis of substrate specificity in malate dehydrogenases: crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase, alpha-ketomalonate and tetrahydoNAD

The structural basis for the extreme discrimination achieved by malate dehydrogenases between a variety of closely related substrates encountered within the cell has been difficult to assess because of the lack of an appropriate catalytically competent structure of the enzyme. Here, we have determined the crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase with the alternative substrate alpha-ketomalonate and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide. Both subunits of the dimeric porcine heart, and from the prokaryotes Escherichia coli and Thermus flavus. However, large changes are noted around the active site, where a mobile loop now closes to bring key residues into contact with the substrate. This observation substantiates a postulated mechanism in which the enzyme achieves high levels of substrate discrimination through charge balancing in the active site. As the activated cofactor/substrate complex has a net negative charge, a positive counter-charge is provided by a conserved arginine in the active site loop. The enzyme must, however, also discriminate against smaller substrates, such as pyruvate. The structure shows in the closed (loop down) catalytically competent complex two arginine residues (91 and 97) are driven into close proximity. Without the complimentary, negative charge of the substrate side-chain of oxaloacetate or alpha-ketomalonate, charge repulsion would resist formation production of this catalytically productive conformation, hence minimising the effectiveness of pyruvate as a substrate. By this mechanism, malate dehydrogenase uses charge balancing to achieve fivefold orders of magnitude in discrimination between potential substrates.     

Properties of malate dehydrogenase isolated from Methanospirillum hungatii.

A NADH-linked oxygen-tolerant malate dehydrogenase was purified 270-fold from cell extracts of Methanospirillum hungatii. Inhibitors of the enzyme included ADP, alpha-ketoglutarate, and excess NADH. Inhibition patterns for ADP were competitive with respect to NADH and non-competitive with respect to oxalacetate. Inhibition by alpha-ketoglutarate was non-competitive with oxalacetate as variable substrate and uncompetitive with respect to NADH. alpha-Ketoglutarate is surmised to function as an end-product inhibitor of the enzyme in reactions converting oxalacetate to alpha-ketoglutarate. No enzyme activity was detected in the direction of malate conversion to oxalacetate, in keeping with a strictly biosynthetic function of the enzyme. An analysis of variance of intial rate data fit to sequential and ping-pong equations showed that a sequential mechanism was perferred. The malate dehydrogenase of M. hungatii resembles those of many other bacteria and eucaryotic cells respect to molecular weight (61,700) and reaction mechanism, but may be regulated differently.     

Chloroplast NADP-malate dehydrogenase: structural basis of light-dependent regulation of activity by thiol oxidation and reduction

BACKGROUND: NADP-dependent malate dehydrogenase (EC 1.1.1.82) is a light-activated chloroplast enzyme that functions in the C4 pathway of photosynthesis. The light regulation is believed to be mediated in vivo by thioredoxin-catalyzed reduction and re-oxidation of cystine residues. The rates of reversible activation and inactivation of the enzyme are strongly influenced by the coenzyme substrates that seem to ultimately determine the steady-state extent of activation in vivo. RESULTS: The X-ray structure of the inactive, oxidized enzyme was determined at 2.8 A resolution. The core structure is homologous to AND-dependent malate dehydrogenases. Two surface-exposed and thioredoxin-accessible disulfide bonds are present, one in the N-terminal extension and the other in the C-terminal extension. The C-terminal peptide of the inactive, oxidized enzyme is constrained by its disulfide bond to fold into the active site over NADP+, hydrogen bonding to the catalytic His225 as well as obstructing access of the C4 acid substrate. Two loops flanking the active site, termed the Arg2 and Trp loops, that contain the C4 acid substrate binding residues are prevented from closing by the C-terminal extension. CONCLUSIONS: The structure explains the role of the C-terminal extension in inhibiting activity. The negative C terminus will interact more strongly with the positively charged nicotinamide of NADP+ than NADPH, explaining why the coenzyme-binding affinities of the enzyme differ so markedly from those of all other homologous alpha-hydroxy acid dehydrogenases. NADP+ may also slow dissociation of the C terminus upon reduction, providing a mechanism for the inhibition of activation by NADP+ but not NADPH.     

Role of NADPH in the regulation of NADP-specific isocitrate dehydrogenase from pig heart.

The present results show that the NADP specific isocitrate dehydrogenase from pig heart exhibits a time lag before the reaction rate approaches a constant value at low metal ion concentrations. Addition of NADPH or EDTA to the assay mixture abolished the lag, and will under certain conditions activate the enzyme.The lag time increased with increasing concentrations of isocitrate and decreased with increasing enzyme concentration. The NADP and metal ion concentration affected the lag in a complex manner. At low NADP and isocitrate concentration, the lag was reduced 50% by an NADPH concentration of less than 2 μm. Stopped flow experiments showed that premixing of NADP or NADPH with the enzyme abolished the effect of NADPH on the lag time. NADPH activated the enzyme at high NADP concentrations. This activating effect could be accounted for by removal of substrate inhibition by NADP.Evidence was obtained to show that the effect of NADPH on the activity was caused by binding of the reduced coenzyme to a site separate from the normal coenzyme binding site. Binding of metal ions by the reduced coenzyme is probably of importance as EDTA affects the lag time and activity in a manner similar to NADPH. The NADPH effect seems to be a general property of NADP-linked isocitrate dehydrogenases.     

Quantitation of the interaction between citrate synthase and malate dehydrogenase

Formation of a bienzyme complex of pig heart mitochondrial malate dehydrogenase and citrate synthase in a buffered system is demonstrated by means of a covalently attached fluorescent probe to citrate synthase. Assuming 1:1 stoichiometry of the enzymes in the complex, an apparent dissociation constant of 10(-6) M was calculated from fluorescence anisotropy measurements. The effect of various metabolites on the interaction was tested. NAD+, oxalacetate, citrate, ATP, and L(-)- or D(+)-malate had no effect on the association of the two enzymes, whereas alpha-ketoglutarate increased and NADH decreased it. The interaction of mitochondrial citrate synthase with cytosolic malate dehydrogenase was found to be much weaker, whereas interaction of citrate synthase with another cytosolic enzyme, aldolase, could not be detected. In kinetic experiments, the activation of malate dehydrogenase by citrate synthase was observed. The effect of pyridine nucleotides and alpha-ketoglutarate is discussed in relation to the direction of the metabolic flow of oxalacetate.     

The nature of the substrate inhibition in lactate dehydrogenases as studied by a spin-labeled derivative of NAD.

The formation of the ternary complex of lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from pig heart and skeletal muscle with the adduct of pyruvate to NAD", spin-labeled at N6 was studied by ultraviolet spectroscopy and ESR techniques. According to ultraviolet measurements we found identical binding characteristics for the natural coenzyme and its spin-labeled analog. The rate by which the ESR signal of free spin-labeled NAD+ decreased upon addition of pyruvate to the binary complexes was substantially different in the two isozymes. With the heart type an initial drop followed by a further linear decrease, zero order in the enzyme and coenzyme concentration was observed. In case of the skeletal muscle isozyme no immediate reaction and a first order process occurred. The initial reaction can be attributed to a non-covalent enzyme/spin-labeled NAD+/pyruvate complex with a dissociation constant for pyruvate of 11 +/- 1 mM, thus explaining the well-known substrate inhibition in the heart isozyme above 2 mM pyruvate. The further reaction is then determined by the buffer dependent enolization of pyruvate. In the muscle isozyme formation of the covalent adduct is not assisted by prior binding of pyruvate in a non-covalent ternary complex and therefore the rate depends on the binary complex concentration.     

A comparative study of NAD+ binding sites in dehydrogenases by circular polarization of fluorescence.

The spectra of the circular polarization of luminescence of a number of dehydrogenases with the fluorescent coenzyme nicotinamide-1,-N⁶-ethenoadenine dinucleotide were measured. By use of this technique it is demonstrated that there is a difference in structure between the adenine subsite in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase on the one hand and pig heart lactate dehydrogenase, horse liver alcohol dehydrogenase, beef liver glutamate dehydrogenase, and pig heart malate dehydrogenase on the other hand. It is concluded that the non-co-operative dehydrogenases have similar, if not identical, adenine subsites whereas in glyceraldehyde-3-phosphate dehydrogenase, a strongly co-operative enzyme, a different structure of the adenine subsite has evolved.     

NAD+-malic enzyme. Regulatory properties of the enzyme from Ascaris suum.

The regulatory properties of the NAD-dependent malic enzyme from the mitochondria of Ascaris suum have been studied. The malate saturation curve exhibits sigmoidicity and the degree of this sigmoidicity increases as the pH is increased. Fumarate was the only compound tested that stimulated the enzyme activity, whereas oxalacetate was the most powerful inhibitor. Activation by low levels of fumarate was found to be competitive with malate. It is proposed that this stimulation has physiological significance in controlling the dismutation reaction in the parasite. The branched-chain volatile fatty acid excretion products, tiglate, 2-methylbutanoate, and 2-methylpentanoate, inhibited the enzyme activity and this inhibition was competitive with malate. The Ki values for these compounds are in the physiological range of their concentrations; therefore, it is suggested that they may aid in controlling the malic enzyme activity in vivo. Oxalacetate inhibition of malic enzyme activity was competitive with malate, and the Ki values decreased with an increase in pH. Two alternatives are proposed which could account for the lack of oxalacetate decarboxylation by the ascarid malic enzyme.