Estimation of rate and dissociation constants involving ternary complexes in reactions catalysed by yeast alcohol dehydrogenase.
Stopped-flow studies of oxidation of butan-1-ol and propan-2-ol by NAD(+) in the presence of Phenol Red and large concentrations of yeast alcohol dehydrogenase give no evidence for the participation of a group of pK(a) approx. 7.6 in alcohol binding. Such a group has been implicated in ethanol binding to horse liver alcohol dehydrogenase [Shore, Gutfreund, Brooks, Santiago & Santiago (1974) Biochemistry13, 4185-4190]. The present result supports previous findings based on steady-state kinetic studies with the yeast enzyme. Stopped-flow studies of the yeast alcohol dehydrogenase-catalysed reduction of acetaldehyde by NADH in the presence of ethanol as product inhibitor indicate that the rate-limiting step is NAD(+) release from the enzyme-NAD(+)-ethanol product complex. This finding permits calculation of K(3), the dissociation constant for ethanol from the enzyme-NAD(+)-ethanol complex, by using the product-inhibition data of Dickenson & Dickinson (1978) (Biochem. J.171, 613-627). The calculations show that K(3) varies very little with pH in the range 5.95-8.9, and this agrees with the findings of the stopped-flow experiments described above. Absorption and fluorescence measurements on mixtures of substrates and coenzymes in the presence of high concentrations of alcohol dehydrogenase have been used to estimate values for the ratio [enzyme-NADH-acetaldehyde]/ [enzyme-NAD(+)-ethanol] at equilibrium. The values obtained were in the range 0.11+/-0.04, and this value together with estimates of K(3) was used to provide estimates of values for rate constants and dissociation constants for steps within the catalytic mechanism.
D000433:1-Propanol / Q000378:metabolism; D000079:Acetaldehyde / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000440:Butanols / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D000440:Butanols; D009243:NAD; D000433:1-Propanol; D000429:Alcohol Oxidoreductases; D000079:Acetaldehyde