1. The activity of liver alcohol dehydrogenase with propan-2-ol and butan-2-ol has been confirmed. The activity with the corresponding ketones is small. Initial-rate parameters are reported for the oxidation of these secondary alcohols, and of propan-1-ol and 2-methylpropan-1-ol, and for the reduction of propionaldehyde and 2-methylpropionaldehyde. Substrate inhibition with primary alcohols is also described. 2. The requirements of the Theorell-Chance mechanism are satisfied by the data for all the primary alcohols and aldehydes, but not by the data for the secondary alcohols. A mechanism that provides for dissociation of either coenzyme or substrate from the reactive ternary complex is described, and shown to account for the initial-rate data for both primary and secondary alcohols, and for isotope-exchange results for the former. With primary alcohols, the rapid rate of reaction of the ternary complex, and its small steady-state concentration, result in conformity of initial-rate data to the requirements of the Theorell-Chance mechanisms. With secondary alcohols, the ternary complex reacts more slowly, its steady-state concentration is greater, and therefore dissociation of coenzyme from it is rate-limiting with non-saturating coenzyme concentrations. 3. Substrate inhibition with large concentrations of primary alcohols is attributed to the formation of an abortive complex of enzyme, NADH and alcohol from which NADH dissociates more slowly than from the enzyme-NADH complex. The initial-rate equation is derived for the complete mechanism, which includes a binary enzyme-alcohol complex and alternative pathways for formation of the reactive ternary complex. This mechanism would also provide, under suitable conditions, for substrate activation or substrate inhibition in a two-substrate reaction, according to the relative rates of reaction through the two pathways.

1. The kinetics of oxidation of ethanol, propan-1-ol, butan-1-ol and propan-2-ol by NAD(+) and of reduction of acetaldehyde and butyraldehyde by NADH catalysed by yeast alcohol dehydrogenase were studied. 2. Results for the aldehyde-NADH reactions are consistent with a compulsory-order mechanism with the rate-limiting step being the dissociation of the product enzyme-NAD(+) complex. In contrast the results for the alcohol-NAD(+) reactions indicate that some dissociation of coenzyme from the active enzyme-NAD(+)-alcohol ternary complexes must occur and that the mechanism is not strictly compulsory-order. The rate-limiting step in ethanol oxidation is the dissociation of the product enzyme-NADH complex but with the other alcohols it is probably the catalytic interconversion of ternary complexes. 3. The rate constants describing the combination of NAD(+) and NADH with the enzyme and the dissociations of these coenzymes from binary complexes with the enzyme were measured.

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The kinetics of ethanol oxidation by NAD+, and acetaldehyde and butyraldehyde reduction by NADH, catalysed by yeast alcohol dehydrogenase, were studied in the pH range 4.9--9.9 at 25 degrees C and in the temperature range 14.8--43.5 degrees C at pH 7.05. The kinetics of reduction of acetaldehyde by [4A-2H]NADH at pH 7.05 and pH 8.9 at 25 degrees C were also studied. The results of the kinetic experiments indicate that the mechanism of catalysis, previously proposed on the basis of studies at pH 7.05 and 25 degrees C (Dickinson & Monger, 1973), applies over the wide range of conditions now tested. Values of some of the initial-rate parameters obtained were used to deduce information about the pH- and temperature-dependence of the specific rates of combination of enzyme and coenzymes and of the dissociation of the enzyme--coenzyme compounds. Primary and secondary plots of initial-rate data are deposited as Supplementary Publication SUP 50043 (20 pages) with the British Library (Lending Division), Boston Spa, Wetherby, Yorks. LS23 7BQ, U.K., from whom copies may be obtained under the terms indicated in Biochem. J. (1975) 145, 5.

1. Inactivation of yeast alcohol dehydrogenase by diethyl pyrocarbonate indicates that one histidine residue per enzyme subunit is necessary for enzymic activity. The inactivated enzyme regains its activity over a period of days. . pyrazole, which are characteristic of native enzyme. 3.The rate constant for the reaction of enzyme with diethyl pyrocarbonate has been determined over the pH range 5.5-9. The histidine residue involved has approximately the same pK, as free histidine, but is 10-fold more reactive than free histidine.Alcohol dehydrogenases from yeast and horse liver each possess a histidine residue, five residues removed from a cysteine residue which, on alkylation with iodoacetate or iodoacetamide, results in the total loss of enzymic activity [l]. It is possible that a histidine residue may be important in catalysis by both of these alcohol dehydrogenases. Accordingly, we have studied the reaction of yeast alcohol dehydrogenase with diethyl pyrocarbonate under conditions where this reagent reacts with histidine residues [2]. Other pyridine-nucleotide-dependent dehydrogenases are known to be inactivated by diethyl pyrocarbonate, e.g. pig heart lactate dehydrogenase [3] and ox liver glutamate dehydrogenase [4]. This paper presents studies on the inactivation of yeast alcohol dehydrogenase by diethyl pyrocarbonate, and on some of the properties of the modified enzyme. The interaction of diethyl pyrocarbonate with horse liver alcohol dehydrogenase has also been examined. A preliminary report of some of the work has been published [5]. MATERIALS AND METHODS MaterialsAll solutions were prepared using glass-distilled water. The sodium phosphate and sodium pyrophos-