Yeast Alcohol Dehydrogenase Reaction Research Articles

Integrated Rate Equation Considering Product Inhibition and Its Application to Kinetic Assay of Serum Ethanol

Kinetic assay of serum ethanol was investigated by predicting maximal product absorbance at 340 nm (A(mk)) through fitting to the yeast alcohol dehydrogenase reaction curve with the integrated rate equation, taking into account product inhibition in the presence of semicarbazide. Predicted A(mk) linearly responded to the preset constant of steady-state concentration of acetaldehyde (C(ald)). An exponential correlation function was established between desired C(ald) and putative A(mk) for authentic ethanol. For unknown samples, iterative fitting to reaction curve till preset constant C(ald) and resultant A(mk) satisfied this exponential correlation function yielded A(mk) with variation coefficient <4.3%. Variations in enzyme activity, data range and kinetic parameters showed negligible effects. The recovery was consistent to 100% with resistance to methanol and isopropanol. The upper limit of linear response for A(mk) was about 40 times of the lower limit. These results indicated that this kinetic method was reliable for serum ethanol assays with obvious advantages.

Transition-state structure in the yeast alcohol dehydrogenase reaction: the magnitude of solvent and alpha-secondary hydrogen isotope effects.

Solvent and alpha-secondary isotope effects have been measured in the yeast alcohol dehydrogenase reaction, under conditions of a rate-limiting transfer of hydrogen between coenzyme and substrate. Determination of catalytic constants (at saturating concentrations of substrate and coenzyme) in H2O and D2O as a function of pH(D) has allowed the separation of solvent effects on pKa from kcat: delta pKa = pKD--pKH = 0.02--0.21, kH2O/kD2O = 1.20 +/- 0.09 in the direction of p-methoxybenzyl alcohol oxidation, and kH2O/kD2O = 0.50 +/- 0.05 and 0.58 +/- 0.06 for p-methoxybenzaldehyde reducation by NADH and [4-2H]NADH. The small effect of D2O on pKa, which contrasts with the common observation that delta pKa congruent to 0.4--0.6, is tentatively assigned to ionization of an active-site ZnOH2. The near absence of an isotope effect on kcat in the direction of alcohol oxidation rules out a mechanism involving concerted catalysis by an active-site base of hydride transfer. In the direction of aldehyde reduction, the observation of inverse isotope effects on kcat is concluded to reflect displacement of zinc-bound water by substrate to form an inner-sphere complex, subsequent to the E.S complex. Equilibrium alpha-secondary isotope effects, measured as a frame of reference for kinetic values, indicate KH/KT = 1.33 +/- 0.05 and 1.34 +/- 0.09 for the oxidation of [1(S)-3H]benzyl alcohol and p-methoxy[1(S)-3H]benzyl alcohol, respectively. Kinetic alpha-secondary isotope effects are within experimental error of equilibrium values, kH/kT = 1.34 +/- 0.07 and 1.38 +/- 0.02 for [1(S)-3H]benzyl alcohol and p-methoxy[1(S)-3H]benzyl alcohol oxidation, respectively. The near identity of kinetic and equilibrium alpha-secondary isotope effects in the direction of alcohol oxidation implicates a transition-state structure which resembles aldehyde with regard to bond hybridization properties. This result contrasts sharply with previously reported structure--reactivity correlations, which implicate a transition-state structure resembling alcohol with regard to charge properties. The significance of these findings to the mechanism of NAD(P)H-dependent redox reactions is discussed.

Isotope effects and structure-reactivity correlations in the yeast alcohol dehydrogenase reaction. A study of the enzyme-catalyzed oxidation of aromatic alcohols.

Steady-state kinetic parameters for the yeast alcohol dehydrogenase catalyzed oxidation of a series of parasubstituted benzyl alcohols-1, 1-h2 and -1, 1-d2 by NAD+ are reported. Catalytic constants have been found to be characterized by large deuterium isotope effects: kH/kD=4.8, p-Br; 4.2, p-Cl; 3, 4, p-H; 4, 2, p-CH3; 3, 2, p-CH3O. The observed isotope effects on k(cat)/K(A), K(A), and K(B), where K(A) and K(B) are Michaelis constants for NAD+ and alcohol, indicate a borderline rapid equilibrium-steady-state kinetic mechanism involving the random addition of substrate and coenzyme to enzyme. With the exception of p-CH3 and possible p-CH3O substituted benzyl alcohol, k(cat) is concluded to represent a single, rate-limiting hydrogen transfer step. A multiple linear regression analysis of the combined data for benzaldehyde reduction (Klinman, J.P. (1972), J. Biol. Chem. 247, 7977-7987, expanded to include p-CH(CH3) 2-substituted benzaldehyde) and benzyl alcohol oxidation has been carried out to determine the contribution of electronic, hydrophobic, and steric effects to k(cat) and substrate binding. Benzaldehyde binding is concluded to depend on electronic substituent effects as previously reported [log 1/K(ald)=(-0.92 +/- 0.18)sigma+-(0.80 +/- 0.067)], whereas benzyl alcohol binding correlates with substrate hydrophobicity [(log 1/K(alc)=(0.60 +/- 0.14) log P -(1.2 +/- 0.12)]. In the case of benzyl alcohol oxidation, k(cat) is independent of electronic and steric effects; the best of seven equations indicates a small negative dependence of k(cat) on hydrophobicity, which is within experimental error or zero [log k(o)=(-0.075 +/- 0.25) log P -(0.65 +/- 0.19)]. Data for benzaldehyde reduction are correlated at the 99% significance level by a single variable equation [(log k(R)=(2.1 +/- 0.37) sigma+-(0.093 +/- 0.14)] and a two variable equation [(log k(R)=(1.9 +/- 0.33) sigma+ + (0.46 +/- 0.20) log P-(0.46 +/- 0.20)]; these equations indicate (a) a large dependence on electronic substituent as reported previously and (b) a possible role for hydrophobic factors in facilitating catalysis. As the result of the observed hydrophobic substituent effects, different ground-state interactions are suggested for the binding of benzaldehydes and benzyl alcohols. Electronic substituent effects lead to the conclusion that there is little or no change in charge at C-1 of substrate at the transition state, relative to alcohol in the ground state. The significance of these effects to the detailed properties of the hydrogen transfer step is discussed.

Acid-base catalysis in the yeast alcohol dehydrogenase reaction.

The effect of pH on steady state kinetic parameters for the yeast alcohol dehydrogenase-catalyzed reduction of aldehydes and oxidation of alcohols has been studied. The oxidation of p-CH3 benzyl alcohol-1,1-h2 and -1,1-d2 by NAD+ was found to be characterized by large deuterium isotope effects (kH/kD = 4.1 plus or minus 0.1) between pH 7.5 and 9.5, indicating a rate-limiting hydride trahsfer step in this pH range; a plot of kCAT versus pH could be fit to a theoretical titration curve, pK = 8.25, where kCAT increases with increasing pH. The Michaelis constnat for p-CH3 benzyl alcohol was independent of pH. The reduction of p-CH3 benzaldehyde by NADH and reduced nicotinamide adenine dinucleotide with deuterium in the 4-A position (NADD) cound not be studied below pH 8.5 due to substrate inhibition; however, between pH 8.5 and 9.5, kCAT was found to decrease with increasing pH and to be characterized by significant isotope effects (kH/kD = 3.3 plus or minus 0.3). In the case of acetaldehyde reduction by NADH and NADD, isotope effects were found to be small and exxentially invariant (kH/kD = 2.O plus or minus 0.4) between pH 7.2 and 9.5, suggesting a partially rate-limiting hydride transger step for this substrate; a plot of kCAT/K'b (where K'b is the Michaelis constant for acetaldehyde) versus pH could be fit to a titration curve, pK = 8.25. The titration curve for acetaldehyde reduction has the same pK but is opposite in direction to that observed for p-CH3 benzyl alcohol oxidation. The data presented in this paper indicate a dependence on different enzyme forms for aldehyde reduction and alcohol oxidation and are consistent with a single active site side chain, pK = 8.25, which functions in acid-base catalysis of the hydride transfer step.

Insolubilized coenzymes: The covalent coupling of enzymatically active NAD to glass surfaces

NAD was attached to the surface of glass beads by a diazo coupling procedure. The insolubilized NAD was shown to function as a coenzyme in the yeast alcohol dehydrogenase reaction.

Nicotinamide-adenine dinucleotide pyrophosphatase in the growing and aging mosquito.

1. The disappearance of pyridine nucleotides during incubation with mosquito homogenates proceeds through the hydrolysis of the pyrophosphate linkage of these compounds as demonstrated by the formation of NMN and AMP from NAD(+). This reaction was also demonstrated by the loss in the coenzyme functioning property of NAD(+) (yeast alcohol dehydrogenase reaction) without a concomitant loss in reactivity towards cyanide. Transglycosidase activity was not observed in the mosquito homogenates, and low concentrations of nicotinamide did not inhibit the NAD(+) splitting activity of these homogenates. These observations are all in accord with the presence in these homogenates of a NAD(+) pyrophosphatase rather than a NADase. 2. The NAD(+) pyrophosphatase is destroyed by boiling, is not heat-activated, and has a pH optimum at pH8.75. In addition to NAD(+), other dinucleotides such as NADP(+), the 3-acetylpyridine and thionicotinamide analogues of NAD(+) and the thionicotinamide analogue of NADP(+), function as substrates in the hydrolysis catalysed by the pyrophosphatase. 3. A decrease in the specific activity of NAD(+) pyrophosphatase was observed during larval development, and a barely detectable activity was found in the pupa and adult. 4. Enzyme activity per organism increased in the larva but decreased to a very low value in the pupa and adult. These results indicate that the decrease in specific activity was due to a decrease in enzyme concentration rather than an increase in amounts of protein.

Mechanisms of Enzyme-catalyzed Oxidation-Reduction Reactions. I. An Investigation of the Yeast Alcohol Dehydrogenase Reaction by Means of the Isotope Rate Effect1,2

Mechanisms of Enzyme-catalyzed Oxidation-Reduction Reactions. I. An Investigation of the Yeast Alcohol Dehydrogenase Reaction by Means of the Isotope Rate Effect^1,2

Yeast alcohol dehydrogenase: I. The effect of pyridine derivatives on the reaction

The effect of pyridine derivatives on yeast alcohol dehydrogenase has been described. These compounds have been found to compete with DPN or DPNH for the active site on the enzyme. Three classes of compounds have been studied: pyridine bases, N 1-methylpyridinium salts, and analogues of DPN. A number of pyridine compounds have been found to show inhibitory powers different for the DPN reduction than for the DPNH oxidation. The inhibition of pyridine bases appears to be due to their ionized forms. Although pyridine bases inhibit the yeast alcohol dehydrogenase reaction, some of these compounds could actually, at low concentrations, stimulate the rate of reaction. A partial reaction sequence and an enzyme-coenzyme model are proposed to explain the experimental data.

Effect of adenine nucleotides on reactions involving triphosphopyridine nucleotide

Oxidized TPN, and P-ADPR have been found to be competitive inhibitors of the TPNH-cytochrome c reductase of horse liver. 2′ AMP also competitively inhibits, at somewhat higher concentrations. DPN, ADPR, desamino TPN, 3′ AMP, 5′ AMP, 2′ IMP, and 3′ IMP produce very slight or no inhibition. Glucose-6-phosphate dehydrogenase (Zwischenferment) is inhibited by a concentration of 3·10 −3 M 2′ AMP, whereas little inhibition results with either 3′ or 5′ AMP. It has been found that DPN when present at high concentrations will react with the yeast phosphogluconic acid dehydrogenase system. Cysteine has been found to activate this enzyme particularly when DPN is used as coenzyme. 2′ AMP inhibits the reduction of both DPN and TPN; the inhibition is much greater when DPN is the electron acceptor. 2′ AMP competitively inhibits the TPN isocitric dehydrogenase from pig heart. 3′ AMP also inhibits but to a lesser degree. The cyclic 2′–3′ AMP, 5′ AMP, and 2′ IMP do not inhibit. 2′ AMP has been found to inhibit the TPN specific isocitrate dehydrogenases from pig heart and brewers yeast, but is without effect on the activity of the DPN specific isocitric dehydrogenases from these sources. Magnesium ions cause a considerable decrease in the inhibition produced by 2′ AMP on the TPN isocitrate system. A similar effect of Mg as well as Mn is observed with the “malic enzyme” from pigeon liver. Both 2′ and 3′ AMP inhibits OAA decarboxylation in pigeon liver; this inhibition requires the addition of metal. TPN stimulation of OAA decarboxylation has been confirmed. TPN reverses the inhibition produced by 3′ AMP, but is less effective against 2′ AMP. None of the adenylic acid isomers at a concentration of 0.01 M influence the yeast alcohol dehydrogenase reaction. 2′ AMP also does not inhibit the oxidation of TPNH by lactic dehydrogenase. The only TPN specific enzyme effect which has been found not to be inhibited by 2′ AMP is the glutathione reductase from peas. The results are discussed with respect to the significance of the monoester phosphate group of TPN in the reactions of this coenzyme.