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-

Stopped-flow studies of oxidation of butan-l-ol and propan-2-ol by NAD+

1. Produced inhibition by ethanol of the acetaldehyde-NADH reaction, catalysed by the alcohol dehydrogenases from yeast and horse liver, was studied at 25 degrees C and pH 6-9. 2. The results with yeast alcohol dehydrogenase are generally consistent with the preferred-pathway mechanism proposed previously [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. The observed hyperbolic inhibition by ethanol of the maximum rate of acetaldehyde reduction confirms the existence of the alternative pathway involving an enzyme-ethanol complex. 3. The maximum rate of acetaldehyde reduction with horse liver alcohol dehydrogenase is also subject to hyperbolic inhibition by ethanol. 4. The measured inhibition constants for ethanol provide some of the information required in the determination of the dissociation constant for ethanol from the active ternary complex. 5. Product inhibition by acetaldehyde of the ethanol-NAD+ reaction with yeast alcohol dehydrogenase was examined briefly. The results are consistent with the proposed mechanism. However, the nature of the inhibition of the maximum rate cannot be determined within the accessible range of experimental conditions. 6. Inhibition of yeast alcohol dehydrogenase by trifluoroethanol was studied at 25 degrees C and pH 6-10. The inhibition was competitive with respect to ethanol in the ethanol-NAD+ reaction. Estimates were made of the dissociation constant for trifluoroethanol from the enzyme-NAD+-trifluoroethanol complex in the range pH6-10.

The half-life for the conversion of malt dimethyl sulphide (DMS) precursor to free DMS has been determined at various temperatures and pH values. At pH 5-2 the half-life of the precursor in wort (S.G. 1 -060) at its boiling point is 38 min, and is doubled for each 6°C fall in temperature. At pH 55 the half-life at the boiling point is 32 5 min.Knowing the stability of the precursor at the various temperatures in the brewing process, the extent of conversion to free DMS in wort at pitching can be predicted for malt of a given precursor content and for a given set of process conditions. The results of DMS analyses of samples taken during brewery trials are in reasonable agreement with the predicted values. This work involved infusion mashing only, but the same principles apply to decoction mashing.The fate of precursor and free DMS during fermentation and conditioning has been followed on a production scale. With some brews, where high levels of free DMS were present at pitching, much free DMS was lost during fermentation. Also, precursor DMS reappeared in the beer after a few days and there was some increase in the level of free DMS.The DMS precursor in green malt has been isolated by ion-exchange and gel-filtration chromatography. A preparation has been obtained which has 0-6 mol potential DMS per mol amino nitrogen. Thin layer chromatography showed that the preparation and its hydrolysis product had the same Rf values as S-methylmethionine and homoserine respectively.