Macroscopic fluctuations in the alcohol dehydrogenase reaction

When studying alcohol dehydrogenase reaction spontaneous oscillations of reaction rate were revealed. The value of these fluctuations essentially exceeded measuring error. The fluctuation amplitude had the maximum value when minimum buffer concentrations were used. Distribution of fluctuations according to their values was polymodal in all experiments. These oscillations seem to be classified as "macroscopic fluctuations" which have been revealed by S. Shnoll & coll [3].     

Anion effects on the liver alcohol dehydrogenase reaction

The effects of various anions on the rate constant for dissociation of NADH from a binary complex with horse liver alcohol dehydrogenase were evaluated. Phosphate, sulfate, and fluoride had no effect, while nitrate and the other halide ions caused a three- to fourfold increase in the rate constant for NADH dissociation. These results indicate that a ternary enzyme-NADH-anion complex is formed, and from the anion concentration dependence the relative affinities are iodide greater than nitrate and bromide greater than chloride. At high salt concentrations, above 0.2 M, the rate constants for NADH dissociation decreased, which was attributed to a decrease in the activity coefficient of the reactants or "salting in." The rate constant for NADH dissociation from ternary complex with imidazole, which crystallizes in an orthorhombic form rather than triclinic, was also substantially enhanced by anions. This provides an indication that the enhancement is independent of the conformational state of the enzyme complex. Thus, the most likely explanation for the observed enhancement of NADH dissociation is anion interference with binding of the coenzyme pyrophosphate group, which does not occur with larger anions such as phosphate or sulfate. Since NADH dissociation partially limits the turnover of the enzyme, the effect of nitrate on steady-state turnover was determined. A twofold increase was observed at optimal levels of nitrate, at both substrate inhibitory and noninhibitory concentrations of ethanol.     

Kinetics of the reaction catalysed by rape alcohol dehydrogenase

The kinetics of the enzyme reaction of ethanol oxidation and acetaldehyde reduction catalysed by alcohol dehydrogenase (ADH) (EC 1.1.1.1) isolated from germinating rape seeds obeys the bi-bi ordered mechanism of Theorell and Chance. The enzyme reaction depends on the pH and temperature. The K_m values for the basic substrates have the lowest values around the pH optimum of the reaction. The enzyme is most stable at pH 6.5–7. The K_m values for ethanol and NAD increase with increasing temperature. The maximum rate of the ethanol oxidation satisfies the Arrhenius equation. The activation energy for the given temperature range is 40.11 kJ/mol. The rape ADH is denatured by heating above 60° but the enzyme-NAD complex is thermally more stable than the enzyme alone.     

Redox-elimination reaction catalyzed by yeast alcohol dehydrogenase

Yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzed reduction of N,N-dimethyl-4-nitrosoaniline by NADH. The stoichiometry of reaction, steady-state kinetic parameters, and the pH-profile for this reaction were estimated. On that basis, the minimal mechanism of the above reaction was postulated.     

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.     

Effect of pH on the liver alcohol dehydrogenase reaction.

New transient kinetic methods, which allow kinetics to be carried out under conditions of excess substrate, have been employed to investigate the kinetics of hydride transfer from NADH to aromatic aldehydes and from aromatic alcohols to NAD+ as a function of pH. The hydride transfer rate from 4-deuterio-NADH to beta-naphthaldehyde is nearly pH independent from pH 6.0 to pH 9.9; the isotope effect is also pH independent with kappa-H/kappaD congruent to 2.3. Likewise, the rate of oxidation of benzyl alcohol by NAD+ changes little with pH between pH 8.75 and pH 5.9; the isotope effect for this process is between 3.0 and 4.4. Earlier substituent effect studies on the reduction of aromatic aldehydes were consistent with electrophilic catalysis by either zinc or a protonic acid. The pH independence of hydride transfer is consistent with electrophilic catalysis by zinc since such catalysis by protonic acid (with a pK between 6.0 and 10.0) would show strong pH dependence. However, protonic acid catalysis cannot be excluded if the pKa of the acid catalyst in the ternary NADH-E-RCOH complex were smaller than 6.0 or smaller than 10.0. The two kinetic parameters changing significantly with pH are the kinetic binding constant for ternary complex formation with aromatic alcohol and the rate of dissociation of aromatic alcohols from enzyme. This is consistent with base-catalyzed removal of a proton from alcohol substrated and consequent acid catalysis of protonation of a zinc-alcoholate complex. The equilibrium constant for hydride transfer from benzaldehyde to benzyl alcohol at pH 8.75 is K-eq equals kappa-H/kappa-H equals 42; this constant has important consequences concerning subunit interactions during liver alcohol dehydrogenase catalysis.     

The reaction of horse-liver alcohol dehydrogenase with glyoxal.

Horse liver alcohol dehydrogenase was reacted with glyoxal at different pH values ranging from 6.0 to 9.0. At pH 9.0 the enzyme undergoes a rapid activation over the first minutes of reaction, followed by a decline of activity, which reaches 10% of that of the native enzyme. Chemical analysis of the inactivated enzyme after sodium borohydride reduction shows that 11 argi-ine and 11 lysine residues per mole are modified. At pH 7.7 the enzyme activity increases during the first hour of the reaction with glyoxal and then decreases slowly. Chemical analysis shows that 4 arginine and 3 lysine residues per mole are modified in the enzyme at the maximum of activation. At pH 7.0 the enzyme undergoes a 4-fold activation. Chemical analysis shows that in this activated enzyme 3 lysine and no arginine residues per mole have been modified. Steady-state kinetic analysis suggests that the activated enzyme is not subjected to substrate inhibition and that its Michaelis constant for ethanol is three times larger than that of the native enzyme. The possible role of arginine and lysine residues in the catalytic function of liver alcohol dehydrogenase is discussed.     

Selective histochemical localization of alcohol dehydrogenase in bud primordia cells of wheat shoot apices

During the histochemical investigation of dehydrogenases in the developing shoot apex of wheat plants it was found that the morphologically similar cells of the peripheral meristem may be differentiated according to the differential activity of alcohol dehydrogenase. While the enzyme was not present or exhibited a very small activity only in the tissue of leaf primordia it was highly active in the cells of bud primordia irrespective of the degree of their development and their differential physiological determination.