Properties of a fructose-1,6-diphosphate-activated lactate dehydrogenase from Acholeplasma laidlawii type A

Acholeplasma laidlawii A possesses a nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactate dehydrogenase (LDH) which is activated specifically by low concentrations of fructose-1, 6-diphosphate (FDP). Studies with partially purified enzyme show that the kinetic response to FDP is hyperbolic. The enzyme is inhibited by inorganic phosphate, adenosine triphosphate, and high concentrations of reduced NAD (NADH). Low activity is demonstrable in the absence of FDP at pH 6.0 to 7.2, but FDP is absolutely required in the region of pH 8. FDP causes an upward shift in the optimum pH of the enzyme, which is near 7.2 in tris (hydroxymethyl)aminomethane buffer. Activation of the enzyme by FDP is markedly affected by substrate concentration; FDP lowers the apparent K(m) for pyruvate and NADH. The affinity of the enzyme for pyruvate is also influenced by H(+) concentration. The pyruvate analogue alpha-ketobutyrate serves as an effective substrate for the enzyme; when it is utilized, the enzyme is still activated by FDP. Reversal of the pyruvate reduction reaction catalyzed by the enzyme can be demonstrated with the 3-acetylpyridine analogue of NAD. The catalytic properties of the A. laidlawii enzyme and the known FDP-activated LDHs which occur among lactic acid bacteria are discussed.     

Identification of an archaeal 2-hydroxy acid dehydrogenase catalyzing reactions involved in coenzyme biosynthesis in methanoarchaea

Two putative malate dehydrogenase genes, MJ1425 and MJ0490, from Methanococcus jannaschii and one from Methanothermus fervidus were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze pyridine nucleotide-dependent oxidation and reduction reactions of the following alpha-hydroxy-alpha-keto acid pairs: (S)-sulfolactic acid and sulfopyruvic acid; (S)-alpha-hydroxyglutaric acid and alpha-ketoglutaric acid; (S)-lactic acid and pyruvic acid; and 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid and 1-oxo-1,3,4, 6-hexanetetracarboxylic acid. Each of these reactions is involved in the formation of coenzyme M, methanopterin, coenzyme F(420), and methanofuran, respectively. Both the MJ1425-encoded enzyme and the MJ0490-encoded enzyme were found to function to different degrees as malate dehydrogenases, reducing oxalacetate to (S)-malate using either NADH or NADPH as a reductant. Both enzymes were found to use either NADH or NADPH to reduce sulfopyruvate to (S)-sulfolactate, but the V(max)/K(m) value for the reduction of sulfopyruvate by NADH using the MJ1425-encoded enzyme was 20 times greater than any other combination of enzymes and pyridine nucleotides. Both the M. fervidus and the MJ1425-encoded enzyme catalyzed the NAD(+)-dependent oxidation of (S)-sulfolactate to sulfopyruvate. The MJ1425-encoded enzyme also catalyzed the NADH-dependent reduction of alpha-ketoglutaric acid to (S)-hydroxyglutaric acid, a component of methanopterin. Neither of the enzymes reduced pyruvate to (S)-lactate, a component of coenzyme F(420). Only the MJ1425-encoded enzyme was found to reduce 1-oxo-1,3,4,6-hexanetetracarboxylic acid, and this reduction occurred only to a small extent and produced an isomer of 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid that is not involved in the biosynthesis of methanofuran c. We conclude that the MJ1425-encoded enzyme is likely to be involved in the biosynthesis of both coenzyme M and methanopterin.     

Production of racemic lactic acid in Pediococcus cerevisiae cultures by two lactate dehydrogenases.

Nicotinamide adenine dinucleotide (NAD)-dependent d(minus)-and l(plus)-lactate dehydrogenases have been partially purified 89- and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for d(minus)-lactate dehydrogenase and 136,000 to 139,000 for l(plus)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by d(minus)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, l(plus)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey Michaelis-Menten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with increased affinity for both substrates at the acid pH. alpha-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevisiae.     

The kinetic mechanism of pyruvate reduction by lactate dehydrogenase from Phycomyces blakesleeanus

The kinetics of pyruvate reduction by lactate dehydrogenase from Phycomyces blakesleeanus NRRL 1555 (-) have been determined at pH 6.0. Initial rate studies performed in the pyruvate reduction direction suggest that a sequential mechanism is operating. Product inhibition studies with NAD+ and L(+)-lactate are consistent with an ordered sequential mechanism if we considered that NAD+ mimics the NADH that binds cooperatively on the enzyme and also the existence of dead-end complex responsible for substrate inhibition by pyruvate at this pH value.     

In situ behaviour of D(-)-lactate dehydrogenase from Escherichia coli

Some kinetic properties of the D(-)-lactate dehydrogenase (EC 1.1.1.28) of Escherichia coli have been investigated. There were marked differences between the kinetic properties of the enzyme studied in situ compared with the in vitro D(-)-lactate dehydrogenase. D(-)-Lactate dehydrogenase in situ showed high substrate inhibition with pyruvate over the pH range 6.0–7.0, whereas the enzyme in vitro did not. The pH optimum for pyruvate reduction by the in situ D(-)-lactate dehydrogenase ranged between pH 7.5 and 7.8, whereas the in vitro enzyme showed its pH optimum between pH 6.8 and 7.0. The pK values of the prototropic groups that controlled the enzymatic activity shift to the acidic region for the in vitro enzyme with respect to the in situ enzyme. In vitro D(-)-lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate and its coenzyme, NADH, at pH values ranging between pH 6.0 and 8.5, but the in situ enzyme showed homotropic interactions neither with pyruvate nor with NADH at all pH values studied.     

Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae.

The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD+ oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saccharomyces cerevisiae strain H44-3D by affinity- and ion-exchange chromatography. SDS-PAGE indicated that the enzyme had a molecular mass of approximately 42,000 (+/- 1,000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer. The pH optimum for the reduction of dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pI of 7.4. NADPH will not substitute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Km values obtained were 0.023 and 0.54 mM for NADH and DHAP, respectively. NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity. Ki values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.     

Lactate dehydrogenase in Phycomyces blakesleeanus.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.     

Characterization of the fructose 1,6-bisphosphate-activated, L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus.

The L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus wt was purified to a final specific activity of 598 mumol pyruvate reduced per min per mg of protein. The specific activity of the pure enzyme with L(+)-lactate was 0.79 units per mg of protein. The M(r) of the native enzyme was 134,000 containing a single subunit type of M(r) 33,500 indicating an apparent tetrameric structure. The L(+)-lactate dehydrogenase was activated by fructose 1,6-bisphosphate in a cooperative manner affecting Vmax and Km values. The activity of the enzyme was also effected by pH, pyruvate and NADH. The Km for NADH at pH 6.0 was 0.05 mM and the Vmax for pyruvate reduction at pH 6.0 was 1082 units per mg in the presence of 1 mM fructose 1,6-bisphosphate. The enzyme was inhibited by NADPH, displaying an uncompetitive pattern. This pattern indicated that NADPH was a negative modifier of the enzyme. The role of L(+)-lactate dehydrogenase in controlling the end products of fermentation is discussed.     

Hymenolepis microstoma: lactate and malate dehydrogenases of the adult worm.

Lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37, respectively) were precipitated with ammonium sulfate, redissolved in 100 mM phosphate buffer, and the kinetic parameters of each enzyme determined. Lactate dehydrogenase: The enzyme preparation had a specific activity of 0.35 μmole NADH oxidized/min/mg protein for pyruvate reduction, and 0.10 μmole NAD reduced/min/mg protein for lactate oxidation. K_m values for the substrates and cofactors were as follows: pyruvate = 0.51, mM; lactate = 3.8 mM; NADH = 0.011 mM; and NAD = 0.17 mM. NADPH, NADP, or d(?)-lactate would not replace NADH, NAD, or l(+)-lactate, respectively. The enzyme was relatively stable at 50 C for 45 min, but much less stable at 60 C; repeated freezing and thawing of the enzyme preparation had little effect on LDH activity. Both p-chloromercuribenzoate (p-CMB) and N-ethylmaleimide (NEM) significantly inhibited LDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least two LDH isoenzymes in the unpurified enzyme preparation. The molecular weight was estimated at 160,000 by gel chromatography. Malate dehydrogenase: The enzyme preparation had a specific activity of 6.70 μmole NADH oxidized/min/mg protein for oxaloacetate reduction, and 0.52 μmole NAD reduced/ min/mg protein for malate oxidation. K_m values for substrates and cofactors were as follows: l-malate = 1.09 mM; oxaloacetate = 0.0059 mM; NADH = 0.017 mM; and NAD = 0.180 mM. NADP and NADPH would not replace NAD and NADH, respectively, d-malate was oxidized slowly when present in high concentrations (>100 mM). Significant substrate inhibition occurred with concentrations of l-malate and oxaloacetate above 40 mM and 0.5 mM, respectively. The enzyme was unstable at temperatures above 40 C, but repeated freezing and thawing of the enzyme preparation had little effect on MDH activity. Only p-CMB inhibited MDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least three MDH isoenzymes in the unpurified enzyme preparation, and the molecular weight was estimated at 49,000 by gel chromatography.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Purification and properties of beta-hydroxybutyrate dehydrogenase from Mycobacterium phlei ATCC354.

beta-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) was purified 145-fold from Mycobacterium phlei ATCC354 by ammonium sulphate fractionation and DEAE-cellulose chromatography. The pH optima for oxidation and reduction reactions were 8.4 and 6.8 respectively. The purified enzyme was specific for NAD, NADH, acetoacetate and D(-)-beta-hydroxybutyrate. Km values for DL-beta-hydroxybutyrate and NAD were 7.4 mM and 0.66 mM respectively. The enzyme was inactivated by mercurial thiol inhibitors and by heat, but could be protected by NADH, Ca2+ and partially by Mn2+. The enzyme did not require metal ions and was insensitive to EDTA, glutathione, dithiothreitol, beta-mercaptoethanol and cysteine.     

Molecular mechanism of VanHst, an alpha-ketoacid dehydrogenase required for glycopeptide antibiotic resistance from a glycopeptide producing organism.

The vancomycin resistance enzyme VanH is an alpha-ketoacid dehydrogenase that stereospecifically reduces pyruvate to D-lactate, which is required for the synthesis of the depsipeptide D-alanine-D-lactate. This compound then forms an integral part of the bacterial cell wall replacing the vancomycin target dipeptide D-alanine-D-alanine, thus the presence of VanH is essential for glycopeptide resistance. In this work, the VanH homologue from the glycopeptide antibiotic producing organism Streptomyces toyocaensis NRRL 15009, VanHst, has been overexpressed in Escherichia coli and purified, and its substrate specificity and mechanism were probed by steady-state kinetic methods and site-directed mutagenesis. The enzyme is highly efficient at pyruvate reduction with kcat/Km = 1.3 x 10(5) M-1 s-1 and has a more restricted alpha-ketoacid substrate specificity than VanH from vancomycin resistant enterococci (VRE). Conversely, VanHst shows no preference between NADH and NADPH while VanH from VRE prefers NADPH. The kinetic mechanism for VanHst was determined using product and dead-end inhibitors to be ordered BiBi with NADH binding first followed by pyruvate and products leaving in the order D-lactate, NAD+. Site-directed mutagenesis indicated that Arg237 plays a role in pyruvate binding and catalysis and that His298 is a candidate for an active-site proton donor. Glu266, which has been suggested to modulate the pKa of the catalytic His in other D-lactate dehydrogenases, was found to fulfill a similar role in VanHst, lowering a pKa value of kcat/Km nearly 2 units. These results now provide the framework for additional structure and inhibitor design work on the VanH family of antibiotic resistance enzymes.     

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

A method is described for the partial purification of pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) cells. The most highly purified preparation catalyzes the reduction of 300 mumoles of nicotinamide adenine dinucleotide (NAD(+)) per min per mg of protein under the assay conditions employed. The enzyme catalyzes the reduction of NAD(+), deamino-NAD(+), and thio-NAD(+) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donor, and the reduction of nicotinamide adenine dinucleotide phosphate (NADP(+)) and thio-NAD(+) with reduced NAD (NADH) as hydrogen donor. The reduction of acetylpyridine AD(+), pyridinealdehyde AD(+), acetylpyridine deamino AD(+), and pyridinealdehydedeamino AD(+) with NADPH as hydrogen donor was not catalyzed. The enzyme catalyzes the transfer of hydrogen more readily from NADPH than from NADH with different hydrogen acceptors. The transfer of hydrogen from NADH to NADP(+) and thio-NAD(+) was markedly stimulated by 2'-adenosine monophosphate (2'-AMP) and inhibited by adenosine diphosphate (ADP), adenosine triphosphate (ATP), and phosphate ions. The transfer of hydrogen from NADPH to NAD(+) was only slightly affected by phosphate ions and 2'-AMP, except at very high concentrations of the latter reagent. In addition, the transfer of hydrogen from NADPH to thio-NAD(+) was only slightly influenced by 2'-AMP, ADP, ATP, and other nucleotides. The kinetics of the transhydrogenase reactions which utilized thio-NAD(+) as hydrogen acceptor and NADH or NADPH as hydrogen donor were studied in some detail. The results suggest that there are distinct binding sites for NADH and NAD(+) and perhaps a third regulator site for NADP(+) or 2'-AMP. The heats of activation for the transhydrogenase reactions were determined. The properties of this enzyme are compared with those of other partially purified transhydrogenases with respect to the regulatory functions of 2'-AMP and other nucleotides on the direction of flow of hydrogen between NAD(+) and NADP(+).     

Purification and characterization of thermostable H<Subscript>2</Subscript>O<Subscript>2</Subscript>-forming NADH oxidase from 2-phenylethanol-assimilating <Emphasis Type="Italic">Brevibacterium</Emphasis> sp. KU1309

A cytoplasmic NADH oxidase (NOX) was purified from a soil bacteria, Brevibacterium sp. KU1309, which is able to grow in the medium containing 2-phenylethanol as the sole source of carbon under an aerobic condition. The enzyme catalyzed the oxidation of NADH to NAD+ involving two-electron reduction of O2 to H2O2. The molecular weight of the enzyme was estimated to be 102 kDa by gel filtration and 57 kDa by SDS-PAGE, which indicates that the NOX was a homodimer consisting of a single subunit. The enzyme was stable up to 70 degrees C at a broad range of pH from 7 to 11. The enzyme activity increased about ten-fold with the addition of ammonium salt, while it was inhibited by Zn2+ (39%), Cu2+ (41%), Hg2+ (72%) and Ag+ (37%). The enzyme acts on NADH, but not on NADPH. The regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or L: -phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling.     

Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.     

Combined coenzyme-substrate analogues of various dehydrogenases. Synthesis of (3S)- and (3R)-5-(3-carboxy-3-hydroxypropyl)nicotinamide adenine dinucleotide and their interaction with (S)- and (R)-lactate-specific dehydrogenases.

Two diastereomeric nicotinamide adenine dinucleotide (NAD+) derivatives were synthesized in which the substrates of (S)-and (R)-lactate-specific dehydrogenases are covalently attached via a methylene spacer at position 5 of the nicotinamide ring. The corresponding nicotinamide derivatives were obtained stereospecifically by enzymatic reduction of 5-(2-oxalylethyl)nicotinamide. (3S)-5-(3-Carboxy-3-hydroxypropyl)-NAD+ undergoes and intramolecular hydride transfer in the presence of pig heart lactate dehydrogenase, forming the corresponding coenzyme-substrate analogue composed of pyruvate and NADH. No cross-reaction products resulting from an intermolecular reaction are observed. Two (R)-lactate specific dehydrogenases, however, do not catalyze a similar reaction in either one of the two diastereomers. A possible arrangement of the substrates in the active centers of these enzymes is proposed. 5-Methyl-NAD+ and 5-methyl-NADH are active coenzymes of pig heart lactate dehydrogenase in contrast to reports in the literature. (S)-Lactate binds to this enzyme in the absence of coenzyme, exhibiting a dissociation constant of 11 mM.     

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.     

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.