Glucose metabolism and dehydrogenase activities in the cytosol and mitochondria of mouse LS cells in chemostat culture

Summary Energy metabolism has been examined in mouse LS cells growing under steady-state conditions in chemostat culture. The metabolic quotient of glucose oxidized (glucose consumed, but not fermented) remained constant, independent of growth rate between cell doubling times of 6 days and 1.2 days. Specific activities of cytochrome oxidase and malate dehydrogenase in the mitochondria remained constant at different growth rates, in accord with the constancy of the glucose oxidation rate. Cytosolic malate dehydrogenase activity was about fourfold greater than the mitochondrial isozyme. The steady-state rate of lactate production fluctuated because of technical limitations but correlated well with cytosolic lactate dehydrogenase activity. This work was supported by Grant A-3458 from the National Sciences and Engineering Research Council Canada.     

Malate dehydrogenase and lactate dehydrogenase in trematodes and turbellarians

Studies have been made on the activity and properties of malate and lactate dehydrogenases from the cattle rumen trematodes Eurytrema pancreaticum, Calicophoron ijimai and the turbellarian Phagocata sibirica which has a common free-living ancestor with the trematodes. All the species studied have a highly active malate dehydrogenase, its activity in the reaction of reducing oxaloacetate being 6-14 times higher than in the reaction of malate oxidation. The affinity of malate dehydrogenase to oxaloacetate was found to be higher than that to malate. The activity of lactate dehydrogenase (reducing the pyruvate) was lower than the activity of malate dehydrogenase, the difference being 50 times for C. ijimai, 4 times for E. pancreaticum and 10 times for P. sibirica.     

Ability of cytosolic malate dehydrogenase and lactate dehydrogenase to increase the ratio of NADPH to NADH oxidation by cytosolic glycerol-3-phosphate dehydrogenase

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, the Vmax of the NADPH oxidation is only slightly lower than the Vmax of NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 microM) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 microM NADH but had no effect on oxidation of 20 microM NADPH by glycerol-3-phosphate dehydrogenase. Consequently malate dehydrogenase increased the ratio of NADPH to NADH oxidation of glycerol-3-phosphate dehydrogenase. On the basis of the measured KD of complexes between malate dehydrogenase and these reduced pyridine nucleotides, and their Km in the glycerol-3-phosphate dehydrogenase reactions, it could be concluded that malate dehydrogenase would have markedly inhibited NADPH oxidation and inhibited NADH oxidation considerably more than observed if its only effect were to decrease the level of free NADH or NADPH. This indicates that due to the opposite chiral specificity of the two enzymes with respect to reduced pyridine nucleotides, complexes between malate dehydrogenase and NADH or NADPH can function as substrates for glycerol-3-phosphate dehydrogenase, but the complex with NADH is less active than free NADH, while the complex with NADPH is as active as free NADPH. Mg2+ enhanced the interactions between malate dehydrogenase and glycerol-3-phosphate dehydrogenase described above. Lactate dehydrogenase (E.C. 1.1.1.27) had effects similar to those of malate dehydrogenase only in the presence of Mg2+. In the absence of Mg2+, there was no evidence of interaction between lactate dehydrogenase and glycerol-3-phosphate dehydrogenase.     

Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity

Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.     

Catalytic properties of dihydroorotate dehydrogenase from Saccharomyces cerevisiae: studies on pH, alternate substrates, and inhibitors

Yeast dihydroorotate dehydrogenase (DHOD) was purified 2800-fold to homogeneity from its natural source. Its sequence is 70% identical to that of the Lactococcus lactis DHOD (family IA) and the two active sites are nearly the same. Incubations of the yeast DHOD with dideuterodihydroorotate (deuterated in the positions eliminated in the dehydrogenation) as the donor and [14C]orotate as the acceptor revealed that the C5 deuteron exchanged with H2O solvent at a rate equal to the 14C exchange rate, whereas the C6 deuteron was infrequently exchanged with H2O solvent, thus indicating that the C6 deuteron of the dihydroorotate is sticky on the flavin cofactor. The pH dependencies of the steady-state parameters (k(cat) and k(cat)/Km) are similar, indicating that k(cat)/Km reports the productive binding of substrate, and the parameters are dependent on the donor-acceptor pair. The lower pKa values for k(cat) and k(cat)/Km observed for substrate dihydroorotate (around 6) in comparison to the values determined for dihydrooxonate (around 8) suggest that the C5 pro S hydrogen atom of dihydroorotate (but not the analogous hydrogen of dihydrooxonate), which is removed in the dehydrogenation, assists in lowering the pKa of the active site base (Cys133). The pH dependencies of the kinetic isotope effects on steady-state parameters observed for the dideuterated dihydroorotate are consistent with the dehydrogenation of substrate being rate limiting at low pH values, with a pKa value approximating that assigned to Cys133. Electron acceptors with dihydroorotate as donor were preferred in the following order: ferricyanide (1), DCPIP (0.54), Qo (0.28), fumarate (0.15), and O2 (0.035). Orotate inhibition profiles versus varied concentrations of dihydroorotate with ferricyanide or O2 as acceptors suggest that both orotate and dihydroorotate have significant affinities for the reduced and oxidized forms of the enzyme.     

The presence of a histidine-aspartic acid pair in the active site of 2-hydroxyacid dehydrogenases. X-ray refinement of cytoplasmic malate dehydrogenase

The structure of cytoplasmic malate dehydrogenase has been partially refined by crystallographic least squares methods. Using x-ray phases based on the refined coordinates, analysis of the resultant electron density maps has led to a new model of cytoplasmic malate dehydrogenase and a tentative "x-ray sequence." The two crystallographically independent subunits comprising the dimeric enzyme are nearly identical in structure and are related to each other by roughly 2-fold rotational symmetry. The best fit of the molecular structure of cytoplasmic malate dehydrogenase to that of lactate dehydrogenase has been obtained by least squares methods. The active sites of these two enzymes contain similarly oriented His-Asp pairs linked by a hydrogen bond which may function as a proton relay system during catalysis. This pair could also provide an explanation for the relatively stronger binding by cytoplasmic malate dehydrogenase and lactate dehydrogenase of NADH versus NAD. Similar His-Asp pairs have been observed in the serine proteases, thermolysin, and phospholipase A2, and the His-Asp pair may play a similar functional role in all of these enzymes.     

A single‐point mutation enables lactate dehydrogenase from Bacillus subtilis to utilize NAD+ and NADP+ as cofactor

The NAD⁺‐dependent lactate dehydrogenase from Bacillus subtilis (BsLDH) catalyzes the enantioselective reduction of pyruvate to lactate. BsLDH is highly specific to NAD⁺ and exhibits only a low activity with NADP⁺ as cofactor. Based on the high activity and good stability of LDHs, these enzymes have been frequently used for the regeneration of NAD⁺. While an application in the regeneration of NADP⁺ is not sufficient due to the cofactor preference of the BsLDH. In addition, NADP⁺‐dependent LDHs have not yet been found in nature. Therefore, a structure‐based approach was performed to predict amino acids involved in the cofactor specificity. Methods of site‐saturation mutagenesis were applied to vary these amino acids, with the aim to alter the cofactor specificity of the BsLDH. Five constructed libraries were screened for improved NADP⁺ acceptance. The mutant V39R was identified to have increased activity with NADP⁺ relative to the wild type. V39R was purified and biochemically characterized. V39R showed excellent kinetic properties with NADP(H) and NAD(H), for instance the maximal specific activity with NADPH was enhanced 100‐fold to 90.8 U/mg. Furthermore, a 249‐fold increased catalytic efficiency was observed. Surprisingly, the activity with NADH was also significantly improved. Overall, we were able to successfully apply V39R in the regeneration of NADP⁺ in an enzyme‐coupled approach combined with the NADP⁺‐dependent alcohol dehydrogenase from Lactobacillus kefir. We demonstrate for the first time an application of an LDH in the regeneration of NADP⁺.     

Crystal structure of the MJ0490 gene product of the hyperthermophilic archaebacterium Methanococcus jannaschii, a novel member of the lactate/malate family of dehydrogenases

The MJ0490 gene, one of the only two genes of Methanococcus jannaschii showing sequence similarity to the lactate/malate family of dehydrogenases, was classified initially as coding for a putative l-lactate dehydrogenase (LDH). It has been re-classified as a malate dehydrogenase (MDH) gene, because it shows significant sequence similarity to MT0188, MDH II from Methanobacterium thermoautotrophicum strain DeltaH. The three-dimensional structure of its gene product has been determined in two crystal forms: a "dimeric" structure in the orthorhombic crystal at 1.9 A resolution and a "tetrameric" structure in the tetragonal crystal at 2.8 A. These structures share a similar subunit fold with other LDHs and MDHs. The tetrameric structure resembles typical tetrameric LDHs. The dimeric structure is equivalent to the P-dimer of tetrameric LDHs, unlike dimeric MDHs, which correspond to the Q-dimer. The structure reveals that the cofactor NADP(H) is bound at the active site, despite the fact that it was not intentionally added during protein purification and crystallization. The preference of NADP(H) over NAD(H) has been supported by activity assays. The cofactor preference is explained by the presence of a glycine residue in the cofactor binding pocket (Gly33), which replaces a conserved aspartate (or glutamate) residue in other NAD-dependent LDHs or MDHs. Preference for NADP(H) is contributed by hydrogen bonds between the oxygen atoms of the monophosphate group and the ribose sugar of adenosine in NADP(H) and the side-chains of Ser9, Arg34, His36, and Ser37. The MDH activity of MJ0490 is made possible by Arg86, which is conserved in MDHs but not in LDHs. The enzymatic assay showed that the MJ0490 protein possesses the fructose-1,6-bisphosphate-activated LDH activity (reduction). Thus the MJ0490 gene product appears to be a novel member of the lactate/malate dehydrogenase family, displaying an LDH scaffold and exhibiting a relaxed substrate and cofactor specificities in NADP(H) and NAD(H)-dependent malate and lactate dehydrogenase reactions.     

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.     

Effect of corticotropin on the dehydrogenase activity of cells and tissues

The authors studied the effect of native ACTH on dehydrogenase activity of isolated strips of rat diaphragm and suspension of E. coli cells, serotype O III:B4, grown on beef extract agar in a medium with different dehydrogenation substrates. ACTH activated dehydrogenase of rat diaphragm in a medium containing pyruvate, alpha-ketoglutarate, malate, beta-hydroxybutyrate, D-aspartic acid and did not alter it in a medium containing succinate. In contradistinction to rat diaphragm, ACTH activated dehydrogenase of E. coli cells whatever the substrates used (oxaloacetate, isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, pyruvate, lactate, beta-hydroxybutyrate, glucose, D-aspartic acid. Synacthen (ACTH1-24) exerted a similar effect. It is suggested that the effects of ACTH are mediated via its influence on adenylate cyclase in the absence of receptors.     

Lactate and malate dehydrogenase binding to the microsomal fraction from chicken liver

Chicken liver microsomal fractions show lactate and malate dehydrogenase activities which behave differently with respect to successive extractions by sonication in 0.15 M NaCl, 0.2% Triton X-100 and 0.15 M NaCl, respectively. The Triton X-100-treated pellet did not show malate dehydrogenase activity but exhibited a 10-fold increase in lactate dehydrogenase activity with respect to the sonicated pellet. Total extracted lactate and malate dehydrogenase activities were, respectively, 7.5 and 1.7 times higher than that in the initial pellet. Different isoenzyme compositions were observed for cytosoluble and microsomal extracted lactate and malate dehydrogenases. When the ionic strength (0-500 mM) or the pH values (6.1-8.7) of the media were increased, an efficient release of lactate dehydrogenase was found at NaCl 30-70 mM and pH 6.6-7.3. Malate dehydrogenase solubilization under the same conditions was very small, even at NaCl 500 mM, but it attained a maximum in the 7.3-8.7 pH range. Cytosoluble lactate dehydrogenase bound in vitro to 0.15 M NaCl-treated (M2) and sonicated (M3) microsomal fractions but not to the crude microsomal fraction (M1). Particle saturation by lactate dehydrogenase occurred with M2 and M3, which contained binding sites with different affinities. Cytosoluble malate dehydrogenase did not bind to M1, M2 and M3 fractions, however, a little binding was found when purified basic malate dehydrogenase was incubated with M2 or M3 fractions.     

Influence of several metabolites on A4 and B4-isoenzymes of loach lactate dehydrogenase activity depending on the direction of the isoenzyme-catalyzed reaction

Various concentration of fructose-1.6-diphosphate, malate, oxaloacetate, creatine phosphate, ATP, ADP and AMP were studied for their effect on the activity of A4-and B4-isoenzymes of lactate dehydrogenase (LDH, EC 1, 1. 1. 27) produced from skeletal muscles and unfertilized egg cells of Misgurnus fossilis in the reactions of lactate oxidation and pyruvate reduction. It was found that oxaloacetate, creatine phosphate, ADP and AMP decreased the activity of A- and B-type isoenzymes to a different extent. The value of the inhibitory action depended not only on the concentration of the substances and subunit composition of the isoenzymes but also depended on the direction of the reaction they catalyse. Malate and fructose-1.6-diphosphate did not inhibit the activity of A4 isoenzyme in the lactate oxidation and malate and ATP did not influence the activity of the former and of B4-isoenzymes in this reaction. At the same time malate, fructose-1.6-diphosphate and ATP decreased the activity of the investigated isoenzymes in the pyruvate reduction reactions.     

Crystal structures of substrate complexes of malic enzyme and insights into the catalytic mechanism

Malic enzymes catalyze the oxidative decarboxylation of L-malate to pyruvate and CO(2) with the reduction of the NAD(P)(+) cofactor in the presence of divalent cations. We report the crystal structures at up to 2.1 A resolution of human mitochondrial NAD(P)(+)-dependent malic enzyme in different pentary complexes with the natural substrate malate or pyruvate, the dinucleotide cofactor NAD(+) or NADH, the divalent cation Mn(2+), and the allosteric activator fumarate. Malate is bound deep in the active site, providing two ligands for the cation, and its C4 carboxylate group is out of plane with the C1-C2-C3 atoms, facilitating decarboxylation. The divalent cation is positioned optimally to catalyze the entire reaction. Lys183 is the general base for the oxidation step, extracting the proton from the C2 hydroxyl of malate. Tyr112-Lys183 functions as the general acid-base pair to catalyze the tautomerization of the enolpyruvate product from decarboxylation to pyruvate.     

The resolution of some steps of the reactions of lactate dehydrogenase with its substrates

1. The reaction of pig heart lactate dehydrogenase (EC 1.1.1.27) with NAD(+) and lactate to form pyruvate and NADH was followed by rapid spectrophotometric methods. The distinct spectrum of enzyme-bound NADH permits the measurement of the rate of dissociation of this compound. 2. The reduction of the first mole equivalent of NAD(+) per mole of enzyme sites can also be observed, and is much more rapid than the steady-state rate of NADH production. 3. At pH8 the dissociation of the enzyme-NADH complex is rate-determining for the steady-state oxidation of lactate. At lower pH some other step after the interconversion of the ternary complex and before the dissociation of NADH is rate-determining. Other evidence for a compulsory-order mechanism is provided.     

Mechanism and applications of phosphite dehydrogenase

Phosphite dehydrogenase catalyzes the NAD+-dependent oxidation of hydrogen phosphonate (common name phosphite) to phosphate in what amounts to a formal phosphoryl transfer reaction from hydride to hydroxide. This review places the enzyme in the context of phosphorus redox metabolism in nature and discusses the results of mechanistic investigations into its reaction mechanism. The potential of the enzyme as a NAD(P)H cofactor regeneration system is discussed as well as efforts to engineer the cofactor specificity of the protein.     

Crystal structure of Escherichia coli malate dehydrogenase. A complex of the apoenzyme and citrate at 1.87 A resolution.

The crystal structure of malate dehydrogenase from Escherichia coli has been determined with a resulting R-factor of 0.187 for X-ray data from 8.0 to 1.87 A. Molecular replacement, using the partially refined structure of porcine mitochondrial malate dehydrogenase as a probe, provided initial phases. The structure of this prokaryotic enzyme is closely homologous with the mitochondrial enzyme but somewhat less similar to cytosolic malate dehydrogenase from eukaryotes. However, all three enzymes are dimeric and form the subunit-subunit interface through similar surface regions. A citrate ion, found in the active site, helps define the residues involved in substrate binding and catalysis. Two arginine residues, R81 and R153, interacting with the citrate are believed to confer substrate specificity. The hydroxyl of the citrate is hydrogen-bonded to a histidine, H177, and similar interactions could be assigned to a bound malate or oxaloacetate. Histidine 177 is also hydrogen-bonded to an aspartate, D150, to form a classic His.Asp pair. Studies of the active site cavity indicate that the bound citrate would occupy part of the site needed for the coenzyme. In a model building study, the cofactor, NAD, was placed into the coenzyme site which exists when the citrate was converted to malate and crystallographic water molecules removed. This hypothetical model of a ternary complex was energy minimized for comparison with the structure of the binary complex of porcine cytosolic malate dehydrogenase. Many residues involved in cofactor binding in the minimized E. coli malate dehydrogenase structure are homologous to coenzyme binding residues in cytosolic malate dehydrogenase. In the energy minimized structure of the ternary complex, the C-4 atom of NAD is in van der Waals' contact with the C-3 atom of the malate. A catalytic cycle involves hydride transfer between these two atoms.     

Inhibition of lactate and malate dehydrogenase by dyes containing vinylsulfone and chlorotriazine groups

The difference spectra of lactate and malate dehydrogenase complexes with four native dyes containing vinylsulfonic and triazinic groups (light-resistant yellow 2KT, red-violet 2KT, etc.) were monitored in 0.1 M phosphate buffer pH 8.2 at 20 degrees C. The dissociation constants were calculated from the spectral data. The most stable complexes were lactate dehydrogenase--light-resistant yellow 2KT and malate dehydrogenase--light-resistant yellow 2KT ones. The values of delta H degree = 5.75 kcal/mole and standard thermodynamic parameters, delta G degree = -6.5 kcal/mole and delta S degree = 41.2 e. u., were calculated from the values of association constants for temperature dependence. The thermodynamic characteristics confirmed the key role of hydrophobic interactions in lactate dehydrogenase--reactive dye complex formation. All the dyes under study competitively inhibit lactate and malate oxidation by the corresponding dehydrogenases. The inhibition constants of both enzymes by the four dyes were determined at 20 degrees C in 0.1 M phosphate buffer pH 8.2. Light-resistant yellow 2KT appeared to be the most effective inhibitor of the enzymes.     

Kinetic analysis of duck epsilon-crystallin with L-lactate dehydrogenase activity: determination of kinetic constants and comparison of substrate specificity.

A systematic analysis of the kinetic properties of duck lens epsilon-crystallin with lactate dehydrogenase [LDH, (E.C. 1.1.1.27)] activity was carried out by employing some 19 different alpha-keto acids as substrates for this NADH-dependent LDH-catalyzed reaction. The steady-state Michaelis and catalytic constants (Km, kcat) were determined for a broad range of organic compounds. The results provide important insights regarding the binding and affinity of substrates to active sites of this enzyme crystallin and indicate a great potential for the application of the stable epsilon-crystallin as a catalyst to the synthesis of some important chiral alpha-hydroxyacids in a convenient and efficient way. It is also demonstrated for the first time that in addition to the enzymatic activity of lactate dehydrogenase, duck epsilon-crystallin also possesses the enzymatic activity of malate dehydrogenase.     

Inhibition of some respiration and dehydrogenase enzyme systems in Escherichia coli NCTC 5933 by phenoxyethanol.

Low concentrations (less than 0.2% w/v) of phenoxyethanol stimulated both the rate of respiration and total oxygen uptakes of Escherichia coli NCTC 5933 suspensions with glucose and other substrates, whilst higher concentrations (0.2--0.6% w/v) although still below those showing significant bactericidal activity, produced progressive levels of inhibition. The degree of respiratory inhibition varied with different substrates in the order malate less than succinate less than pyruvate less than or equal to glucose less than lactate, and suggested appreciable inhibition at a point after malate in the tricarboxylic acid cycle. This suggestion was supported by the use of tetrazolium salts as alternative electron acceptors, and by cytochrome difference spectra, which together implicated malate dehydrogenase as the most likely site of action. Isolated dehydrogenase enzymes of the tricarboxylic acid cycle in cell-free preparations were unaffected by high concentrations of phenoxyethanol (0.8% w/v) with the exception of malate dehydrogenase which was inhibited in extracts to extents similar to those of malate oxidation by intact bacteria. Lineweaver-Burke plots for malate dehydrogenase activity in the presence of phenoxyethanol suggested a competitive inhibition of the oxaloacetic acid-limited reaction and a non-competitive inhibition of the NADH-limited reaction. Accordingly, Ki values were found to be low when the rate of reaction was limited by oxaloacetic acid concentration yet relatively high when NADH was rate limiting.     

An enzymatic cycling method for 3-acetylpyridine adenine dinucleotide to increase the sensitivity of enzymatic methods which employ this NAD analog

The NAD analog 3-acetylpyridine adenine nucleotide (APAD), because of its higher oxidation potential, has proven useful for the direct enzymatic measurement of such compounds as lactate, malate, glutamate, etc., for which the equilibrium with NAD+ as oxidant is unfavorable. An enzymatic cycling method which is capable of increasing the sensitivity of such reactions 10,000-fold or more is described. The APADH produced in the original stoichiometric reaction is used to catalyze a cycling reaction that employs lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37) to generate (from lactate plus oxalacetate) very large quantities of pyruvate and malate. After the cycling step, the malate formed is measured with NAD+ and with malate dehydrogenase, plus aspartate aminotransferase, and oxaloacetate to pull this indicator reaction to completion. The application of this cycling method is illustrated by analysis of malate in the range 1 to 10 pmol.