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PubMed1976

Repression of sporulation in Bacillus subtilis by L-malate.

L-Malate repressed sporulation in the wild-type strain of Bacillus subtilis. When 75 mM L-malate was added to the growth medium at the time of inoculation, the appearance of heat-resistant spores was delayed 6 to 8 h. The synthesis of extracellular serine protease, alkaline phosphatase, glucose dehydrogenase, and dipicolinic acid was similarly delayed. Sporulation was not repressed when malate was added to the culture at t4 or later. A mutant was selected for ability to sporulate in the presence of malate. This strain could also sporulate in the presence of glucose. The malate-resistant mutant grew poorly with malate as sole carbon source, although it possessed an intact citric acid cycle, and it showed increased levels of malic enzyme. This indicates a defect in the metabolism of malate in the mutant. A mutant lacking malate dehydrogenase activity was also able to sporulate in the presence of malate. A model for the regulation of sporulation by malate is presented and discussed. Citric acid cycle intermediates other than malate did not affect sporulation. In contrast to previous results, sporulation of certain citric acid cycle mutants could be greatly increased or completely restored by the addition of intermediates after the enzymatic block. The results indicate that the failure of citric acid cycle mutants to sporulate can be adequately explained by lack of energy and lack of glutamate.

Alcohol Oxidoreductases↗

PubMed1975

De novo synthesis and developmental control of the multiple gene-controlled malate dehydrogenase isozymes in maize scutella.

In maize, both soluble and mitochondrial malate dehydrogenases (L-malate:NAD oxidoreductase, EC 1.1.1.37)exist in multiple molecular forms (isozymes). Biochemical studies and genetic analysis showed that the maize malate dehydrogenase isozymes are not interconvertable conformational forms of a single gene product. Instead, they are determined by multiple genes which may reside on different chromosomes. Therefore, developmental expression of the various malate dehydrogenase isoqymes was studied. During early development of the sporophyte (dry kernel to 10 days of germination), the total malate dehydrogenase activity in scutella increases through the first 5 days, peaks about the 6th day and decreases gradually thereafter. Both soluble and mitochondrial malate dehydrogenase isozymes exhibit similar activity profiles; however, the total mitochondrial malate dehydro genase activity is only 60% of that in the cytosol. Density labeling experiments and atempts to detect the possible existence of "inactive malate dehydrogenase precursors" were performed. The results show that both soluble and mitochondrial malate dehydrogenase are synthesized in the scutella during germination and early growth of young maize seedlings. Accumulation of the maize malate dehydrogenase isozymes is probably controlled by synthesis as well as degradation instead of activation and inactivation of enzyme moieties. Correlation between the developmental pattern of the scutellar malate dehydrogenase isozymes and the physiological conditions of the young maize seedlings is discussed.

Centrifugation, Density Gradient↗

PubMed2004

Carbohydrate metabolism in two apple genotypes that differ in malate accumulation.

In the apple variety 'Usterapfel', there are two known genotypes, which differ in malic acid content. One hundred days after full bloom, low-acid fruit (LA-fruit) contained 125 micromolg(-1) dry matter (DW) of malate, while the high-acid genotype (HA-fruit) reached levels up to 627 micromolg(-1) DW. There was no difference in the catalytic activity of enzymes involved in malate metabolism, such as PEPcarboxylase, malate dehydrogenase, and NADP malic enzyme. After [14C]glucose incorporation into the excised tissue of either genotype, the organic acid fraction was labeled to approximately the same extent. Furthermore, uptake of [14C]malate was significantly lower in excised tissue of LA-fruit. These findings suggest that low malate content in LA-fruit is the result of a restricted ability to accumulate malate in apple parenchyma cells. The different ability to accumulate malate had a pronounced effect on overall carbon partitioning. However, the rate of respiration and the rate of malate synthesis was similar in both genotypes. In HA-fruit, the glycolytic flux through pyruvate kinase was increased to compensate for the carbon that accumulated in the vacuole as malate. Since malate storage in the LA-fruit was restricted, it was more easily available for gluconeogenesis, and was correlated with a three-times higher activity of PEPcarboxykinase. LA-fruit showed higher concentrations of ATP, which stimulated Glc6P and fructose-6-phosphate formation. The elevated hexosephosphate content led to an enhanced partitioning of carbon into starch (+40%), hemicellulose (+104%), and sucrose (+40%) in more mature fruit. The activation of carbohydrate synthesis resulted in a significant drop in glucose-1-phosphate (Glc1P). To meet the increased demand for Glc1P, the activities of neutral and acid invertase, hexokinase, and phosphoglucomutase were higher in LA-fruit. Glucose was a more versatile substrate for this metabolic route than was fructose. It was also evident that glycolytic flux in apple was dependent on glucose level, and that the reaction catalysed by phosphoglucomutase contributed to the regulation of carbon partitioning between malate and carbohydrate polymers.

Carbohydrate Metabolism↗

PubMed2002

Tartrate dehydrogenase catalyzes the stepwise oxidative decarboxylation of D-malate with both NAD and thio-NAD.

Tartrate dehydrogenase catalyzes the divalent metal ion- and NAD-dependent oxidative decarboxylation of D-malate to yield CO(2), pyruvate, and NADH. The enzyme also catalyzes the metal ion-dependent oxidation of (+)-tartrate to yield oxaloglycolate and NADH. pH-rate profiles and isotope effects were measured to probe the mechanism of this unique enzyme. Data suggest a general base mechanism with likely general acid catalysis in the oxidative decarboxylation of D-malate. Of interest, the mechanism of oxidative decarboxylation of D-malate is stepwise with NAD(+) or the more oxidizing thio-NAD(+). The mechanism does not become concerted with the latter as observed for the malic enzyme, which catalyzes the oxidative decarboxylation of L-malate [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096-2103]. It appears the change in mechanism observed with malic enzyme is specific to its transition state structure and not a generalized trait of metal ion- and NAD(P)-dependent beta-hydroxy acid oxidative decarboxylases. The V/K(malate) pH-rate profile decreases at low and high pH and exhibits pK(a) values of about 6.3 and 8.3, while that for V/K(tartrate) (measured from pH 7.5 to pH 9) exhibits a pK(a) of 8.6 on the basic side. A single pK(a) of 6.3 is observed on the acid side of the V(max) pH profile, but the pK(a) seen on the basic side of the V/K pH profiles is not observed in the V(max) pH profiles. Data suggest the requirement for a general base that accepts a proton from the 2-hydroxyl group of either substrate to facilitate hydride transfer. A second enzymatic group is also required protonated for optimum binding of substrates and may also function as a general acid to donate a proton to the enolpyruvate intermediate to form pyruvate. The (13)C isotope effect, measured on the decarboxylation of D-malate using NAD(+) as the dinucleotide substrate, decreases from a value of 1.0096 +/- 0.0006 with D-malate to 1.00787 +/- 0.00006 with D-malate-2-d, suggesting a stepwise mechanism for the oxidative decarboxylation of D-malate. Using thio-NAD(+) as the dinucleotide substrate the (13)C isotope effects are 1.0034 +/- 0.0007 and 1.0027 +/- 0.0002 with D-malate and D-malate-2-d, respectively.

Alcohol Oxidoreductases↗

PubMed1971

Metabolism of pyruvate and malate by isolated fat-cell mitochondria.

1. Metabolism of pyruvate and malate by isolated fat-cell mitochondria incubated in the presence of ADP and phosphate has been studied by measuring rates of pyruvate uptake, malate utilization or production, citrate production and oxygen consumption. From these measurements calculations of the flow rates through pyruvate carboxylase, pyruvate dehydrogenase and citrate cycle have been made under various conditions. 2. In the presence of bicarbonate, pyruvate was largely converted into citrate and malate and only about 10% was oxidized by the citrate cycle; citrate and malate outputs were linear after lag periods of 6-9min and 3min respectively, and no other end products of pyruvate metabolism were detected. On the further addition of malate or hydroxymalonate, the lag in the rate of citrate output was less marked but no net malate disappearance was detected. If, however, bicarbonate was omitted then net malate uptake was observed. Addition of butyl malonate was found to greatly inhibit the metabolism of pyruvate to citrate and malate in the presence of bicarbonate. 3. These results are in agreement with earlier conclusions that in adipose tissue acetyl units for fatty acid synthesis are transferred to the cytoplasm as citrate and that this transfer requires malate presumably for counter transport. They also support the view that oxaloacetate for citrate synthesis is preferentially formed from pyruvate through pyruvate carboxylase rather than malate through malate dehydrogenase and that the mitochondrial metabolism of citrate in fat-cells is restricted. The possible consequences of these conclusions are discussed. 4. Studies on the effects of additions of adenine nucleotides to pyruvate metabolism by isolated fat-cell mitochondria are consistent with inhibition of pyruvate carboxylase in the presence of ADP and pyruvate dehydrogenase in the presence of ATP.

Adenosine Diphosphate↗

PubMed1998

Interaction between citrate synthase and malate dehydrogenase. Substrate channeling of oxaloacetate.

The interactions between pig heart citrate synthase and mitochondrial malate dehydrogenase or cytosolic malate dehydrogenase were studied using the frontal analysis method of gel filtration and by precipitation in polyethylene glycol. This method showed that an interaction between citrate synthase and mitochondrial malate dehydrogenase occurred but no interaction between citrate synthase and cytosolic malate dehydrogenase. Channeling of oxaloacetate in the malate dehydrogenase and citrate synthase-coupled systems was tested using polyethylene glycol precipitates of citrate synthase and mitochondrial malate dehydrogenase, and citrate synthase and cytosolic malate dehydrogenase. The effectiveness of large amounts of aspartate aminotransferase and oxaloacetate decarboxylase, as competing enzymes for the intermediate oxaloacetate, was examined. Aspartate aminotransferase and oxaloacetate decarboxylase were less effective competitors for oxaloacetate when precipitated citrate synthase and mitochondrial malate dehydrogenase in polyethylene glycol was used at low ionic strength compared with free enzymes in the absence of polyethylene glycol or with a co-precipitate of citrate synthase and cytosolic malate dehydrogenase. Substrate channeling of oxaloacetate with citrate synthase-mitochondrial malate dehydrogenase precipitate was inefficient at high ionic strength. These effects could be explained through electrostatic interactions of mitochondrial but not cytosolic malate dehydrogenase with citrate synthase.

Animals↗

PubMed1980

19S cytosolic malate synthase. A small pool characterized by rapid turnover.

A pool of 19S malate synthase was detected in the cytosol. This pool was separated from the microsomal and glyoxysomal malate synthase when cotyledons of germinating seed of Cucumis sativus were fractionated. An early stage of seed germination was selected for our investigations, when 100S "microsomal" malate synthase was also present. 1) When L-[35S]methionine was applied in vivo to label cellular proteins the small pool of 19S malate synthase was found to contain the highest specific activity compared to microsomal or glyoxysomal malate synthase. 2) The kinetics of specific radioactivity in malate synthase during a pulse chase-labelling experiment established that 19S malate synthase was a precursor of microsomal malate synthase. 3) By means of anti-malate synthase antibodies, 100S malate synthase could be recovered on protein A-Sepharose and thus separated from the endoplasmic reticulum. The data suggest that both microsomal and glyoxysomal malate synthase are synthesized in the cytosol rather than at the endoplasmic reticulum according to the signal hypothesis.

Cytosol↗

PubMed1981

The immobilization of mitochondrial malate dehydrogenase on Sepharose beads and the demonstration of catalytically active subunits.

Porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been immobilized by covalent attachment to CNBr-activated Sepharose 4B-Cl gel. The gel was activated with low levels of CNBr to produce a low density of linkage sites and, hence, to facilitate linkage of the enzyme through a single subunit. Matrix-bound mitochondrial malate dehydrogenase was found to possess 50-65% of the native mitochondrial malate dehydrogenase specific activity when assayed in the NAD+ leads to NADH direction but only 5-15% of the native enzyme specific activity when assayed in the NADH leads to NAD+ direction. MB-dimeric mitochondrial malate dehydrogenase was dissociated to MB-monomer by exposure to pH 5.0 buffer. The MB-monomer was found to be catalytically active, possessing only a slightly decreased specific activity when compared to MB-dimer. The reconstitution of Mb-monomer to MB-dimer was accomplished by adding dissociated mitochondrial malate dehydrogenase, which exists at pH 5.0, to MB-monomer and adjusting to pH 7.5. The kinetic parameters, pH activity profile, and stability toward heat denaturation for MB-mitochondrial malate dehydrogenase (monomer and dimer) were determined and compared to native mitochondrial malate dehydrogenase. MB-mitochondrial malate dehydrogenase exhibited enhanced stability and similar pH activity profiles when compared to native mitochondrial malate dehydrogenase. Immobilization of mitochondrial malate dehydrogenase altered the enzyme's kinetic parameters in such a manner as to increase the values of Km for the substrates and decrease the values of Vmax.

Animals↗

PubMed1974

An apparent oligomer of malate dehydrogenase from bean leaves.

Two forms of malate dehydrogenase of widely differing molecular weight have been examined from primary leaves of Phaseolus vulgaris. In addition to the normal 69,000 molecular weight enzyme, an unusual form of 280,000 molecular weight may be detected by sucrose density gradient centrifugation or gel filtration with Sephadex G-200. Isopycnic density gradient centrifugation showed that both forms of malate dehydrogenase differed markedly from the bulk of the leaf protein by their low bouyant density of 1.261 g/cm(3).High molecular weight (280,000) malate dehydrogenase could be converted to active low molecular weight (69,000) malate dehydrogenase by treatment with 2.5 m CsCl, 1.0 m NaCl, 6 m urea, pH 6.5 or below, or one freeze-thaw cycle. Simple removal of salt or raising the pH were not effective in reforming the high molecular weight malate dehydrogenase after dissociation. The high molecular weight enzyme was not dissociated during prolonged dialysis against 0.1 m NaCl or 0.05 m phosphate, pH 7.0. Calcium at concentrations up to 0.1 m produced no activation or differential response in the two MDH forms.High and low molecular weight malate dehydrogenase were nearly identical in susceptibility to inhibition by various unreactive substrate analogs. However, there was a marked difference in the ability of the two forms of malate dehydrogenase to reduce 3-acetylpyridine-deamino-NAD. This difference in activity was the basis of a convenient assay for determining the ratio of high to low molecular weight malate dehydrogenase in crude extracts. The pH activity profiles and Michaelis constant for malate were nearly identical for the two molecular weight forms.Analysis by polyacrylamide gel electrophoresis revealed one high molecular weight and two low molecular weight malate dehydrogenase zones. Dissociation of high molecular weight malate dehydrogenase resulted in formation of low molecular weight enzyme whose electrophoretic properties differed from the normal low molecular weight forms.

Journal Article↗

PubMed1982

Determination of NAD Malic Enzyme in Leaves of C(4) Plants : EFFECTS OF MALATE DEHYDROGENASE AND OTHER FACTORS.

Malate dehydrogenase may interfere with the assay of NAD malic enzyme, as NADH is formed during the conversion of malate to oxaloacetate. During the present study, two additional effects of malate dehydrogenase were investigated; they are evident only if the malate dehydrogenase reaction is allowed to reach equilibrium prior to initiating the malic enzyme reaction. One of these (Outlaw, Manchester 1980 Plant Physiol 65: 1136-1138) might cause an underestimation of NAD reduction by malic enzyme due to the oxidation of NADH during reversal of the malate dehydrogenase reaction. A second effect may result in overestimation of malic enzyme activity, as Mn(2+)-catalyzed oxaloacetate decarboxylation causes continuing net NADH formation via malate dehydrogenase. These effects were studied by assaying the activity of a partially purified preparation of Amaranthus retroflexus NAD malic enzyme in the presence or absence of purified NAD malate dehydrogenase.A model was developed which allowed the generation of theoretical curves describing the influence of malate dehydrogenase on the assay of malic enzyme activity. The experimental data obtained agreed closely with these curves. The conditions included 5 millimolar malate, 2 millimolar NAD, and 4 millimolar Mn(2+) (pH 7.2 to 7.8 at 30 degrees C). At low activities of malic enzyme (1 nanomole per minute per milliliter or less), the presence of malate dehydrogenase leads to a substantial overestimation of malic enzyme activity due to the Mn(2+)-catalyzed decarboxylation of oxaloacetate having a dominant effect. When the level of malic enzyme is greater than 1 nanomole per minute per milliliter, reversal of malate dehydrogenase has a dominant transient effect, causing a lag of up to several minutes, after which the change in absorbance reflects the true rate of malic enzyme. Independent of this effect is a lag in the activator-dependent rate, which could be eliminated by preincubating the enzyme with activator (coenzyme A).An assay procedure designed to minimize the influence of these effects is described. New data are presented on the activity of NAD malic enzyme in leaves of different subgroups of C(4) plants.

Journal Article↗

PubMed1975

Variations in the pathways of malate oxidation and phosphorylation in different species of Mycobacteria.

Mycobacterium tuberculosis H37Rv, the slow-growing human pathogenic strain of tubercle bacilli and Mycobacterium smegmatis and Mycobacterium phlei, the fast-growing saprophytes, have shown variations regarding the type of dehydrogenase that initiates malate oxidation in the respiratory chain. M. tuberculosis H37Rv is characterized by having a malate oxidase system (designated MALNAD pathway) in which malate oxidation is mediated by the NAD+-dependent malate dehydrogenase (EC 1.1.1.37) but not by FAD-dependent malate-vitamin K reductase. M. smegmatis possesses a different malate oxidase system (designated MALFAD pathway) in which malate oxidation is exclusively carried out by the FAD-dependent malate-vitamin K reductase because NAD+-dependent malate dehydrogenase is absent in this organism. M. phlei has a mixed system of malate oxidase (designated MALNAD+FAD pathways) in which both the NAD+-and FAD-dependent dehydrogenases take part. In all the three systems, the rest of the electron transport chain is common.

Alcohol Oxidoreductases↗

PubMed1980

Effect of bicarbonate and oxaloacetate on malate oxidation by spinach leaf mitochondria.

Mitochondria isolated from spinach leaves oxidized malate by both a NAD+-linked malic enzyme and malate dehydrogenase. In the presence of sodium arsenite the accumuation of oxaloacetate and pyruvate during malate oxidation was strongly dependent on the malate concentration, the pH in the reaction medium and the metabolic state condition. Bicarbonate, especially at alkaline pH, inhibited the decarboxylation of malate by the NAD+-linked malic enzyme in vitro and in vivo. Analysis of the reaction products showed that with 15 mM bicarbonate, spinach leaf mitochondria excreted almost exclusively oxaloacetate. The inhibition by oxaloacetate of malate oxidation by spinach leaf mitochondria was strongly dependent on malate concentration, the pH in the reaction medium and on the metabolic state condition. The data were interpreted as indicating that: (a) the concentration of oxaloacetate on both sides of the inner mitochondrial membrane governed the efflux and influx of oxaloacetate; (b) the NAD+/NADH ratio played an important role in regulating malate oxidation in plant mitochondria; (c) both enzymes (malate dehydrogenase and NAD+-linked malic enzyme) were competing at the level of the pyridine nucleotide pool, and (d) the NAD+-linked malic enzyme provided NADH for the reversal of the reaction catalyzed by the malate dehydrogenase.

Bicarbonates↗

PubMed2004

Respiratory properties and malate metabolism in Percoll-purified mitochondria isolated from pineapple, Ananas comosus (L.) Merr. cv. smooth cayenne.

An investigation was made of the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant Ananas comosus (pineapple) in malate metabolism during CAM phase III. Pineapple mitochondria showed very high malate dehydrogenase (MDH), and low malic enzyme (ME) and glutamate-oxaloacetate transaminase (GOT) activities. The mitochondria readily oxidized succinate and NADH with high rates and coupling, while they only oxidized NADPH in the presence of Ca(2+). Pineapple mitochondria oxidized malate with low rates under most assay conditions, despite increasing malate concentrations, optimizing pH, providing cofactors such as coenzyme A, thiamine pyrophosphate, and NAD(+), and supplying individually external glutamate or GOT. However, providing glutamate and GOT simultaneously strongly increased the rates of malate oxidation. The OAA easily permeated the mitochondrial membranes to import into or export out of pineapple mitochondria during malate oxidation, but the mitochondria did not consume external Asp or alpha-KG. These results suggest that OAA played a significant role in the mitochondrial malate metabolism of pineapple, in which malate was mainly oxidized by active mMDH to produce OAA which could be exported outside the mitochondria via a malate-OAA shuttle. Cytosolic GOT then consumed OAA by transamination in the presence of glutamate, leading to a large increase in respiration rates. The malate-OAA shuttle might operate as a supporting system for decarboxylation in phase III of PCK-CAM pineapple. This shuttle system may be important in pineapple to provide a source of energy and substrate OAA for cytosolic PCK activity during the day when cytosolic OAA and ATP was limited for the overall decarboxylation process.

Ananas↗

PubMed1975

Purification and properties of malate dehydrogenase from Pseudomonas testosteroni.

Nicotinamide adenine dinucleotide-linked malate dehydrogenase has been purified from Pseudomonas testosteroni (ATCC 11996). The purification represents over 450-fold increase in specific activity. The amino acid composition of the enzyme was determined and found to be quite different from the composition of the malate dehydrogenases from animal sources as well as from Escherichia coli. Despite this difference, however, the data show that the enzymatic properties of the purified enzyme are remarkably similar to those of other malate dehydrogenases that have been previously studied. The Pseudomonas enzyme has a molecular weight of 74,000 and consists of two subunits of identical size. In addition to L-malate, the enzyme slowly oxidizes other four-carbon dicarboylates having an alpha-hydroxyl group of S configuration such as meso- and (-) tartrate. Rate-determining steps, which differ from that of the reaction involving L-malate, are discussed for the reaction involving these alternative substrates. Oxidation of hydroxymalonate, a process previously undetected with other malate dehydrogenases, is demonstrated fluorometrically. Hydroxymalonate and D-malate strongly enhance the fluorescence of the reduced nicotinamide adenine dinucleotide bound to the enzyme. The enzyme is A-stereospecific with respect to the coenzyme. Malate dehydrogenase is present in a single form in the Pseudomonas. The susceptibility of the enzyme to activation or inhibition by its substrates-particularly the favoring of the oxidation of malate at elevated concentrations-strongly resembles the properties of the mitochondrial enzymes. The present study reveals that whereas profound variations in chemical composition have occurred between the prokaryotic and eukaryotic enzymes, the physical and catalytic properties of malate dehydrogenase, unlike lactate dehydrogenase, are well conserved during the evolutionary process.

Amino Acids↗

PubMed1969

L-malate oxidation by the electron transport fraction of Azotobacter vinelandii.

The membrane-bound l-malate oxidoreductase of Azotobacter vinelandii strain O was found to be a flavoprotein-dependent enzyme associated with the electron transport system (R(3)) of this organism. The particulate R(3) fraction, which possessed the l-malate oxidoreductase, carried out the cyanide-sensitive oxidation of l-malate, d-lactate, reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, succinate, cytochrome c, tetramethyl-p-phenylenediamine, and p-phenylenediamine, with molecular O(2) as the terminal electron acceptor. d-Malate was not oxidized, but l-malate was oxidized to oxalacetate. Phenazine methosulfate (PMS), vitamin K(3), K(3)Fe(CN)(6), nitro blue tetrazolium, and dichloroindophenol all served as good terminal electron acceptors for the l-malate oxidoreductase. Cytochrome c was a poor electron acceptor. Extensive studies on the l-malate oxidase and PMS and K(3) reductases revealed that all were stimulated specifically by flavine adenine dinucleotide and nonspecifically by di- or trivalent cations, i.e., Ca(++), Ba(++), Mn(++), Mg(++), Fe(+++), Ni(++), and Al(+++). All these activities were markedly sensitive to ethylenediaminetetraacetate (EDTA). The V(max) values for the l-malate oxidase, PMS, and vitamin K(3) reductases were, respectively, 3.4, 15.1, and 45.5 mumoles of substrate oxidized per min per mg of protein at 37 C. Spectral studies revealed that the Azotobacter R(3) flavoprotein and cytochromes (a(2), a(1), b(1), c(4), and c(5)) were reduced by l-malate. l-Malate oxidase activity was sensitive to various inhibitors of the electron transport system, namely, p-chloromercuriphenylsulfonic acid, chlorpromazine, 2-n-heptyl-4-hydroxyquinoline-N-oxide, antimycin A, and KCN. Minor inhibitory effects were noted with the inhibitors 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione, rotenone, and Amytal.

Antimycin A↗

PubMed1984

The glucose-dependent transport of L-malate in Zygosaccharomyces bailii.

Zygosaccharomyces bailii possesses a constitutive malic enzyme, but only small amounts of malate are decomposed when the cells ferment fructose. Cells growing anaerobically on glucose (glucose cells) decompose malate, whereas fructose cells do not. Only glucose cells show an increase in the intracellular concentration of malate when suspended in a malate-containing solution. The transport system for malate is induced by glucose, but it is repressed by fructose. The synthesis of this transport system is inhibited by cycloheximide. Of the two enantiomers L-malate is transported preferentially. The transport of malate by induced cells is not only inhibited by addition of fructose but also inactivated. This inactivation is independent of the presence of cycloheximide. The transport of malate is inhibited by uranyl ions; various other inhibitors of transport and phosphorylation were of little influence. It is assumed that the inducible protein carrier for malate operates by facilitated diffusion. Fructose cells of Z. bailii and cells of Saccharomyces cerevisiae do not contain a transport system for malate.

Biological Transport↗

PubMed1991

Rat liver mitochondrial malate dehydrogenase: purification, kinetic properties, and role in ethanol metabolism.

Malate dehydrogenase was purified from the mitochondrial fraction of rat liver by ion-exchange chromatography with affinity elution. The kinetic parameters for the enzyme were determined at pH 7.4 and 37 degrees C, yielding the following values (microM): Ka, 72; Kia, 11; Kb, 110; Kp, 1600; Kip, 7100; Kq, 170; Kiq, 1100, where a = NADH, b = oxalacetate, p = malate, and q = NAD+. Kib was estimated to be about 100 microM. The maximum velocities for mitochondrial malate dehydrogenase in rat liver homogenates, at pH 7.4 and 37 degrees C, were 380 +/- 40 mumol/min per gram of liver, wet weight, for oxalacetate reduction and 39 +/- 3 mumol/min per gram of liver, wet weight, for malate oxidation. Rates of the reaction catalyzed by mitochondrial malate dehydrogenase under conditions similar to those in vivo were calculated using these kinetic parameters and were much lower than the maximum velocity of the enzyme. Since mitochondrial malate dehydrogenase is not saturated with malate at physiological concentrations, its kinetic parameters are probably important in the regulation of mitochondrial malate concentration during ethanol metabolism. For the mitochondrial enzyme to operate at a rate comparable to the flux through cytosolic malate dehydrogenase during ethanol metabolism (about 4 mumol min-1 per gram liver), the mitochondrial [malate] would need to be about 2 mM and the mitochondrial [oxalacetate] would need to be less than 1 microM.

Animals↗

PubMed1998

How fumarase recycles after the malate --> fumarate reaction. Insights into the reaction mechanism.

Recycling of yeast fumarase to permit repetition of its reaction chemistry requires two proton transfers and two conformational changes, in pathways that are different in detail but thematically similar in the two directions. In the malate --> fumarate direction, simple anions such as acetate accelerate the fumarate-off step producing E(H(f)), a fumarate-specific isoform that retains the C3R-proton of malate. Fumarate specificity is shown with S-2,3-dicarboxyaziridine, which is competitive vs fumarate and noncompetitive with malate as substrate. The steady-state level of E(H(f)), based on Kii (S-2,3-dicarboxyaziridine), is increased by D2O and decreased by imidazole acting as a general acid for conversion of E(H(f)) to E(H(f))H. E(H(f))H is fumarate-specific as shown by the inhibition pattern with ClO4-. The pKa of this step is approximately 7.25 based on the pH dependence of Kii (ClO4-). A conformational change occurs next as shown by high sensitivity of k(cat) but not k(cat)/Km, to the microviscosogen, glycerol, and change to a nonspecific isoform, E(H(mf))H, probably the same species formed in the fumarate --> malate direction from malate-specific intermediates by a different conformational change. Malate enters the cycle by reaction with E(H(mf))H and returns to E(m)H x malate after a second conformational change. When fumarate-off is slow, as in low anion medium, malate itself becomes an activator of malate --> fumarate. This effect occurs with changes in inhibition patterns suggestive of the bypass of the slow E(f) --> E(mf) conversion in favor of direct formation of E(mf) when free fumarate is formed. 3-Nitro-2-hydroxypropionate, a strong inhibitor of fumarase [Porter, D. J. T., and Bright, H. J. (1980) J. Biol. Chem. 255, 4772-4780] in its carbanion form, is competitive with both malate and fumarate. Therefore, 3-nitro-2-hydroxypropionic acid interacts with E(H(mf))H and not with E(m) or E(f) isoforms. Occurrence of two different conformational changes in the recycling process suggests that the reaction chemistry employs a two-step mechanism. The specificity of inhibition for E(H(mf))H is consistent with the expected intermediate of a carbanion mechanism, E(H)H x carbanion-. The proton transfers and conformational changes of recycling occur in the same sequence that is expected for this reaction chemistry. Several examples of ligand-activated conformational changes are reported.

Animals↗