Human glutamic-gamma-semialdehyde dehydrogenase. Kinetic mechanism.

The kinetic mechanism of homogeneous human glutamic-gamma-semialdehyde dehydrogenase (EC 1.5.1.12) with glutamic gamma-semialdehyde as substrate was determined by initial-velocity, product-inhibition and dead-end-inhibition studies to be compulsory ordered with rapid interconversion of the ternary complexes (Theorell-Chance). Product-inhibition studies with NADH gave a competitive pattern versus varied NAD+ concentrations and a non-competitive pattern versus varied glutamic gamma-semialdehyde concentrations, whereas those with glutamate gave a competitive pattern versus varied glutamic gamma-semialdehyde concentrations and a non-competitive pattern versus varied NAD+ concentrations. The order of substrate binding and release was determined by dead-end-inhibition studies with ADP-ribose and L-proline as the inhibitors and shown to be: NAD+ binds to the enzyme first, followed by glutamic gamma-semialdehyde, with glutamic acid being released before NADH. The Kia and Kib values were 15 +/- 7 microM and 12.5 microM respectively, and the Ka and Kb values were 374 +/- 40 microM and 316 +/- 36 microM respectively; the maximal velocity V was 70 +/- 5 mumol of NADH/min per mg of enzyme. Both NADH and glutamate were product inhibitors, with Ki values of 63 microM and 15,200 microM respectively. NADH release from the enzyme may be the rate-limiting step for the overall reaction.     

Modification of mouse testicular lactate dehydrogenase by pyridoxal 5''-phosphate.

1. Mouse C4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.7 and 25 degrees C loses activity gradually; 1mM-pyridoxal 5'-phosphate causes 83% inactivation, and higher concentrations of the reagent cause no further loss of activity. 2. The final extent of inactivation is very pH-dependent, greater inactivation occurring at the high pH values. 3. Inactivation may be fully reversed by addition of cysteine, or made permanent by reducing the enzyme with NaBH4. 4. The absorption spectrum of inactivated reduced enzyme indicates modification of lysine residues. Inactivation by 80% corresponds to modification of at least 1.8 mol of lysine/mol of enzyme subunit. 5. There is no loss of free thiol groups after inactivation with pyridoxal 5'-phosphate and reduction of the enzyme. 6. NAD+ or NADH gives complete protection against inactivation. protection studies with coenzyme fragments indicate that the AMP moiety is largely responsible for the protective effect. Lactate (10 mM) gives no protection in the absence of added nucleotides, but greatly enhances the protection given by ADP-ribose (1 mM). Thus ADP-ribose is able to trigger the binding of lactate. 7. Pyridoxal 5'-phosphate also acts as a non-covalent inhibitor of mouse C4 lactate dehydrogenase. The inhibition is non-competitive with respect to both NAD+ and lactate. 8. Km values for the enzyme at pH 8.0 and 25 degrees C, with the non-varied substrate saturating, are 0.3 mM-lactate and 5 microM-NAD+. 9. These results are discussed and compared with pyridoxal 5'-phosphate modification of other lactate dehydrogenase isoenzymes and related dehydrogenases.     

Mannitol-1-phosphate dehydrogenase of Escherichia coli. Chemical properties and binding of substrates.

Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5'-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5'-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.     

D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate.

Steady-state kinetic studies including initial velocity for mannitol oxidation and fructose reduction and product inhibition for mannitol oxidation using fructose and reduced nicotinamide adenine dinucleotide (NADH) are in accord with a reaction mechanism best described as ordered Bi-Bi with NAD+ and NADH designated as the first substrate, last product, respectively at pH 8.8. All replots of slopes and intercepts from product inhibition studies were linear. Dead-end inhibition studies using mannitol 1-phosphate gave slope-parabolic, intercept-linear noncompetitive inhibition for both NAD+ and mannitol as substrates. The dead-end inhibitor is capable of binding multiply to the E, EA, and EQ forms of the enzyme to an extent that is controlled by the concentration of substrates. The EQ complex is inferred to undergo a conformational change, E'Q equilibrium EQ, since (V1/E1) greater than (KiqV2)/(KqE1), and no evidence for dead-end complex formation with NADH can be adduced. This is interpreted to mean that the release of fructose from the central complex is faster than the isomerization of the E-NADH complex. When mannitol is saturating, the noncompetitive inhibition against NAD+, as the variable substrate, becomes parabolic uncompetitive. A replot of the slopes of the parabola against mannitol 1-phosphate remains concave upward. This situation could arise if the conformational change we infer in the EQ complex opens up additional sites on the protein which can interact with the dead-end inhibitor.     

The reactions of 1,10-phenanthroline with yeast alcohol dehydrogenase.

Freshly prepared samples of yeast alcohol dehydrogenase (EC 1.1.1.1) were inhibited by 1,10-phenanthroline at pH 7.0 and 0 degrees C in a two-stage process. The first step appeared to be slowly established, but was rendered reversible by removal of reagent or by addition of excess Zn2+ ions. The second step was irreversible and was associated with the dissociation of the tetrameric enzyme. The presence of saturating concentrations of NAD+ or NADH promoted and enhanced inhibition by the slowly established reversible process, but prevented dissociation of the enzyme. For the incubation mixtures containing NAD+, removal of the 1,10-phenanthroline resulted in virtually complete recovery of activity, whereas, for the incubation mixtures containing NADH, removal of the reagent gave only partial re-activation. The presence of NAD+ and pyrazole, or NADH and acetamide, in incubation mixtures with the enzyme gave rise to ternary complexes that gave protection against both forms of inactivation by 1,10-phenanthroline. The results support the view that at least some of the Zn2+ ions associated with yeast alcohol dehydrogenase have a catalytic, as opposed to a purely structural, role.     

Rates of glucose utilization and glucogenesis in rats in the basal state induced by halothane anaesthesia.

Methyl methanethiosulphonate was used to produce a modification of the essential thiol group in lactate dehydrogenase which leaves the enzyme catalytically active. Methyl methanethiosulphonate produced a progressive inhibition of enzyme activity, with 2mM-pyruvate and 0.14mM-NADH as substrates, which ceased once the enzyme had lost 70-90% of its activity. In contrast, with 10mM-lactate and 0.4mM-NAD+ as substrates the enzyme was virtually completely inhibited. The observed inhibition was critically dependent on the chosen substrate concentration, since methanethiolation with methyl methanethiosulphonate resulted in a large decrease in affinity for pyruvate. At 0.14mM-NADH, methanethiolation increased the apparent KmPyr from from 40micronM for the control enzyme to 12mM for the modified enzyme. Steady-state kinetics showed that there was not a statistically significant change in either KmNADH or KsNADH. At saturating NADH and pyruvate concentrations, the Vmax. was virtually unaffected for the methanethiolated enzyme. However, a decrease in Vmax. was observed when the modified enzyme was incubated in dilute solution. The modification of lactate dehydrogenase by methyl methanethiosulphonate involved the active site, since inhibition was completely prevented by substrate-analogue pairs such as NADH and oxamate or NAD+ and oxalate. The formation of complexes between methanethiolated lactate dehydrogenase and substrates or substrate analogues can also be shown by re-activation experiments. The methanethiolated enzyme was re-activated in a time-dependent reaction by dithiothreitol and this was prevented by oxamate, by NADH and by NADH plus oxamate in increasing order of effectiveness. The results of this work are interpreted in terms of a role for the essential thiol group in the binding of substrates.     

A simple steady state kinetic model for alcohol dehydrogenase. Use of reaction progress curves for parameters estimation at pH 7.4

A simple rate equation for alcohol dehydrogenase was obtained by assuming independent binding sites for ethanol and NAD+ and fully competitive inhibition by the products of the reaction, acetaldehyde and NADH. A random binding order was also assumed. The rate equation is described by six parameters: four association constants (two for the substrates and two for the products of the reaction), Vf for the forward direction, and the equilibrium constant of the reaction. The six parameters were determined at pH 7.4 by numerical analysis of progress curves of reactions started with different concentrations of ethanol and NAD+. The parameters for alcohol dehydrogenase partially purified from rat liver were: Km for ethanol = 0.746 mM, Km for NAD+ = 0.0563 mM, Km for acetaldehyde = 7.07 microM, Km for NADH = 4.77 microM and Keq = 2.36 X 10(-4). The computed values allowed a very good simulation of the experimental progress curves and little variation was observed in the kinetic parameters when the reactions were started in the presence of either NADH or acetaldehyde.     

The inhibition of pepsin-catalysed reactions by products and product analogues. Kinetic evidence for ordered release of products

1. The inhibition of pepsin-catalysed hydrolysis of N-acetyl-l-phenylalanyl-l-phenylalanylglycine by products and product analogues was studied. 2. The non-competitive nature of the inhibition by the product N-acetyl-l-phenylalanine confirms an ordered release of products, and points to a common mechanism (involving an amino-enzyme) for pepsin-catalysed transpeptidation and hydrolysis reactions. 3. N-Acetyl-l-phenylalanine ethyl ester is also a non-competitive inhibitor, but here the inhibition is of the ;dead-end' type. No ethanol is detectable in reaction mixtures, indicating that this ester cannot act as an amino group acceptor in a transpeptidation process. 4. The same is true for N-methanesulphonyl-l-phenylalanine methyl and methyl thiol esters. No methanethiol is liberated when the methyl thiol ester is present as an inhibitor of the hydrolytic reaction, and the hope that such a thiol ester would effectively trap the amino-enzyme was not fulfilled.     

Kinetic mechanism of a flavonol-ring-B O-glucosyltransferase from Chrysosplenium americanum

The kinetic mechanism of a flavonol-ring-B O-glucosyltransferase from Chrysosplenium americanum was investigated. Substrate interaction kinetics for the flavonol and UDPG gave converging lines, which were consistent with a sequential bireactant binding mechanism. They also showed substrate inhibition with respect to the flavonol. Intercept and slope replots were linear and gave a KA of 250 microM and a KB of 10 microM. Product-inhibition studies showed competitive inhibition between UDPG and UDP (KiQ 20 microM) and non-competitive inhibition between the flavonol substrate and its glucoside (KiP 1 mM). Kinetic patterns were consistent with an ordered bi-bi mechanism, where UDPG is the first substrate to bind to the enzyme and UDP is the final product released. The high KiP value, as compared with that of KB, indicates that the reaction is not inhibited by the glucosylated products formed and conforms with the accumulation of flavonol glucosides in C. americanum.     

Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.     

Purification of an inducible L-valine dehydrogenase of Streptomyces coelicolor A3(2)

Valine dehydrogenase (VDH) from Streptomyces coelicolor A3(2) was purified from cell-free extracts to apparent homogeneity. The enzyme had an Mr 41,000 in denaturing conditions and an Mr 70,000 by gel filtration chromatography, indicating that it is composed of two identical subunits. It oxidized L-valine and L-alpha-aminobutyric acid efficiently, L-isoleucine and L-leucine less efficiently, and did not act on D-valine. It required NAD+ as cofactor and could not use NADP+. Maximum dehydrogenase activity with valine was at pH 10.5 and the maximum reductive amination activity with 2-oxoisovaleric acid and NH4Cl was at pH 9. The enzyme exhibited substrate inhibition in the forward direction and a kinetic pattern with NAD+ that was consistent with a sequential ordered mechanism with non-competitive inhibition by valine. The following Michaelis constants were calculated from these data: L-valine, 10.0 mM; NAD+, 0.17 mM; 2-oxoisovalerate, 0.6 mM; and NADH, 0.093 mM. In minimal medium, VDH activity was repressed in the presence of glucose and NH4+, or glycerol and NH4+ or asparagine, and was induced by D- and L-valine. The time required for full induction was about 24 h and the level of induction was 2- to 23-fold.     

The essential active-site lysines of clostridial glutamate dehydrogenase. A study with pyridoxal-5'-phosphate.

Glutamate dehydrogenase (GDH) of Clostridium symbiosum, like GDH from other species, is inactivated by pyridoxal 5'-phosphate (pyridoxal-P). This inactivation follows a similar pattern to that for beef liver GDH, in which a non-covalent GDH-pyridoxal-P complex reacts slowly to form a covalent complex in which pyridoxal-P is in a Schiff's-base linkage to lysine residues. [formula: see text] The equilibrium constant of this first-order reaction on the enzyme surface determines the final extent of inactivation observed [S. S. Chen and P. C. Engel (1975) Biochem. J. 147, 351-358]. For clostridial GDH, the maximal inactivation obtained was about 70%, reached after 10 min with 7 mM pyridoxal-P at pH 7. In keeping with the model, (a) inactivation became irreversible after reduction with NaBH4. (b) The NaBH4-reduced enzyme showed a new absorption peak at 325 nm. (c) Km values for NAD+ and glutamate were unaltered, although Vmax values were decreased by 70%. Kinetic analysis of the inactivation gave values of 0.81 +/- 0.34 min-1 for k3 and 3.61 +/- 0.95 mM for k2/k1. The linear plot of 1/(1-R) against 1/[pyridoxal-P], where R is the limiting residual activity reached in an inactivation reaction, gave a slightly higher value for k2/k1 of 4.8 +/- 0.47 mM and k4 of 0.16 +/- 0.01 min-1. NADH, NAD+, 2-oxoglutarate, glutarate and succinate separately gave partial protection against inactivation, the biggest effect being that of 40 mM succinate (68% activity compared with 33% in the control). Paired combinations of glutarate or 2-oxoglutarate and NAD+ gave slightly better protection than the separate components, but the most effective combination was 40 mM 2-oxoglutarate with 1 mM NADH (85% activity at equilibrium). 70% inactivated enzyme showed an incorporation of 0.7 mM pyridoxal-P/mol subunit, estimated spectrophotometrically after NaBH4 reduction, in keeping with the 1:1 stoichiometry for the inactivation. In a sample protected with 2-oxoglutarate and NADH, however, incorporation was 0.45 mol/mol, as against 0.15 mol/mol expected (85% active). Tryptic peptides of the enzyme, modified with and without protection, were purified by HPLC. Two major peaks containing phosphopyridoxyllysine were unique to the unprotected enzyme. These peaks yielded three peptide sequences clearly homologous to sequences of other GDH species. In each case, a gap at which no obvious phenylthiohydantoin-amino-acid was detected, matched a conserved lysine position. The gap was taken to indicate phosphopyridoxyllysine which had prevented tryptic cleavage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Asparagusate dehydrogenases and lipoyl dehydrogenase from asparagus mitochondria. Physical, chemical, and enzymatic properties.

Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have been obtained in homogeneous state from asparagus mitochondria. They are flavin enzymes with 1 mol of FAD/mol of protein. Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have s20,w of 6.22 S, 6.39 S, and 5.91 S, respectively, and molecular weights of 111,000, 110,000, and 95,000 (sedimentation equilibrium) or 112,000, 112,000, and 92,000 (gel filtration). They are slightly acidic proteins with isoelectric points of 6.75, 5.75, and 6.80. Both asparagusate dehydrogenases catalyzed the reaction Asg(SH)2 + NAD+ equilibrium AsgS2 + NADH + H+ and exhibit lipoyl dehydrogenase and diaphorase activities. Lipoyl dehydrogenase is specific for lipoate and has no asparagusate dehydrogenase activity. NADP cannot replace NAD in any case. Optimum pH for substrate reduction of the three enzymes are near 5.9. Asparagusate dehydrogenases I and II have Km values of 21.5 mM and 20.0 mM for asparagusate and 3.0 mM and 3.3 mM for lipoate, respectively. Lipoyl dehydrogenase activity of asparagusate dehydrogenases is enhanced by NAD and surfactants such as lecithin and Tween 80, but asparagusate dehydrogenase activity is not enhanced. Asparagusate dehydrogenases are strongly inhibited by mercuric ion, p-chloromercuribenzoic acid, and N-ethylmaleimide. Amino acid composition of the three enzymes is presented and discussed.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

Studies on the biosynthesis of cytochrome P-450 in rat liver--a probe with phenobarbital.

The reaction of NAD(P)H:flavin oxidoreductase (flavin reductase) from Photobacterium fischeri is proposed to follow a ping-pong bisubstrate-biproduct mechanism. This is based on a steady-state kinetic analysis of initial velocities and patterns of inhibition by NAD⁺ and AMP. The double reciprocal plots of initial velocities versus concentrations of FMN or NADH show, in both cases, a series of parallel lines. The Michaelis constants for NADH (FMN saturating) and FMN (NADH saturating) are 2.2 and 1.2 × 10^?4m, respectively. The product NAD⁺ has been found to be an inhibitor competitive with FMN but non-competitive with NADH. Using AMP as an inhibitor, noncompetitive inhibition patterns were observed with respect to both NADH and FMN as the varied substrate. In addition, the reductase was not inactivated by treatment with N-ethylmaleimide either alone or in the presence of FMN, but the enzyme was inactivated by N-ethylmaleimide in the presence of NADH. These findings suggest that flavin reductase shuttles between disulfide- and sulfhydryl-containing forms during catalysis.     

Distinction between NAD- and NADH-binding forms of mitochondrial malate dehydrogenase as shown by inhibition with thenoyltrifuoroacetone.

The inhibition of mitochondrial malate dehydrogenase (L-malate : NADH oxidoreductase, EC 1.1.1.37) by 2-thenoyltrifluoroacetone (TTFA) was investigated at pH 8.0 where both forward and backward reactions can be measured. The inhibition with respect to malate is non-competitive at finite NAD concentrations. Increasing the NAD concentrations lowers the slope of the double reciprocal plot so that at infinite NAD the inhibition is uncompetitive. The inhibition with respect to oxaloacetate is non-competitive. Increasing the NADH concentration lowers the slope and intercept of the double reciprocal plot so that at infinite NADH the inhibition is nil. The inhibition with respect to NADH is competitive, whatever the oxaloacetate concentrations are. The inhibition with respect to NAD, at all malate concentrations, is non-competitive. This pattern of inhibition is incompatible with any model assuming that NAD and NADH reacts with identical forms of the enzyme. On the other hand the reciprocating compulsory ordered mechanism, where the two subunits of the dimeric enzyme are working in concert, can account for all the experimental results. It is concluded that NAD and NADH bind to different forms of the enzyme separated by reversible steps. Only one form (see text), the one which binds NADH, can react to form the dead end complex (see text). The similarity between mechanism of inhibition by thenoyltrifluoroacetone and other hydrophobic inhibitors of malate dehydrogenase is discussed.     

Activation of nitrite reductase from Escherichia coli K12 by oxidized nicotinamide-adenine dinucleotide.

Nitrite reductase from Escherichia coli K12 requires the presence of NAD+, one of the products of the reduction of NO2-by NADH, for full activity. The effect is observed with both crude extracts and purified enzyme. NAD+ also acts as a product inhibitor at high concentrations, and plots of initial rate against NAD+ concentration are bell-shaped. The maximum occurs at about 1 mM-NAD+, but increases with increasing NADH concentration. In the presence of 1 mM-NAD+ and saturating NO2-(2mM) the Michaelis constant for NADH is about 16 micron. The Michaelis constant for NO2-is about 5 micron and is largely independent of the NAD+ concentration. Similar but more pronounced effects of NAD+ are observed with hydroxylamine as electron acceptor instead of NO2-. The maximum rate of NADH oxidation by hydroxylamine is about 5.4 times greater than the maximum rate of NADH oxidation by NO2- when assayed with the same volume of the same preparation of purified enzyme. The Michaelis constant for hydroxylamine is 5.3 mM, however, about 1000 times higher than for NO2-. These results are consistent with a mechanism in which the same enzyme-hydroxylamine complex occurs as an intermediate in both reactions.     

A complex effect of arsenite on the formation of alpha-ketoglutarate in rat liver mitochondria

This investigation presents disturbances of the mitochondrial metabolism by arsenite, a hydrophilic dithiol reagent known as an inhibitor of mitochondrial alpha-keto acid dehydrogenases. Arsenite at concentrations of 0.1-1.0 mM was shown to induce a considerable oxidation of intramitochondrial NADPH, NADH, and glutathione without decreasing the mitochondrial membrane potential. The oxidation of NAD(P)H required the presence of phosphate and was sensitive to ruthenium red, but occurred without the addition of calcium salts. Mitochondrial reactions producing alpha-ketoglutarate from glutamate and isocitrate were modulated by arsenite through various mechanisms: (i) both glutamate transaminations, with oxaloacetate and with pyruvate, were inhibited by accumulating alpha-ketoglutarate; however, at low concentrations of alpha-ketoglutarate the aspartate aminotransferase reaction was stimulated due to the increase of NAD+ content; (ii) the oxidation of isocitrate was stimulated at its low concentration only, due to the oxidation of NADPH and NADH; this oxidation was prevented by concentrations of citrate or isocitrate greater than 1 mM; (iii) the conversion of isocitrate to citrate was suppressed, presumably as a result of the decrease of Mg2+ concentration in mitochondria. Thus the depletion of mitochondrial vicinal thiol groups in hydrophilic domains disturbs the mitochondrial metabolism not only by the inhibition of alpha-keto acid dehydrogenases but also by the oxidation of NAD(P)H and, possibly, by the change in the ion concentrations.     

Studies on NAD+ permeability in intact mitochondria from rabbit reticulocytes.

Functionally intact mitochondria from rabbit reticulocytes are characterized by a low NAD+ level after the preparation (0.29 nmoles NAD+ + NADH/mg protein). They are apparently impermeable for NADH and exhibit a slow net uptake of NAD+. From the increase of O2-uptake in state 3 and the increase of NADH concentration in state 4 of respiration after the addition of NAD+ we concluded that 3--10 min are necessary for the saturation with NAD+ at 23 degrees C. 2mM NAD+ extramitochondrially are not sufficient to saturate the mitochondria with NADH and probably NAD+, too. Because of the net uptake of NAD+ we assume that reticulocyte mitochondria lose NAD+ during their preparation. If they are incubated with the physiological concentration of 300 micrometer NAD+, which was found in reticulocytes, a value of 1.9 nmoles NAD+ + NADH mg protein was calculated. At an extramitochondrial NAD+ concentration of 300 micrometer, reticulocyte mitochondria exhibit an almost maximal O2-uptake in the presence of oxaloacetate or alpha-ketoglutarate. It is concluded that the mitochondria in intact reticulocytes contain the "normal" complement of NAD+ + NADH.     

Purification and characterization of a dimeric phenylalanine dehydrogenase from Rhodococcus maris K-18.

NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.) was purified to homogeneity from a crude extract of Rhodococcus maris K-18 isolated from soil. The enzyme had a molecular mass of about 70,000 daltons and consisted of two identical subunits. The enzyme catalyzed the oxidative deamination of L-phenylalanine and several other L-amino acids and the reductive amination of phenylpyruvate and p-hydroxyphenylpyruvate. The enzyme required NAD+ as a natural coenzyme. The NAD+ analog 3-acetylpyridine-NAD+ showed much greater coenzyme activity than did NAD+. D-Phenylalanine, D-tyrosine, and phenylethylamine inhibited the oxidative deamination of L-phenylalanine. The enzyme reaction was inhibited by p-chloromercuribenzoate and HgCl2. Initial-velocity and product inhibition studies showed that the reductive amination proceeded through a sequential ordered ternary-binary mechanism. NADH bound first to the enzyme, followed by phenylpyruvate and then ammonia, and the products were released in the order L-phenylalanine and NAD+. The Michaelis constants were as follows: L-phenylalanine, 3.8 mM; NAD+, 0.25 mM; NADH, 43 microM; phenylpyruvate, 0.50 mM; and ammonia, 70 mM.