YEAST ALCOHOL DEHYDROGENASE: MOLECULAR WEIGHT, COENZYME BINDING, AND REACTION EQUILIBRIA

Abstract

The alcohol dehydrogenases (ADH) crystallized from yeast (1, 2) and from horse liver (3) differ in many of their properties. The mammalian enzyme forms a complex with reduced diphosphopyridine nucleotide in which the absorption band of the coenzyme at 340 rnp is shifted to 325 rnh (4). This permitted the direct study by Theorell and Chance (5) of the stoichiometry and dissociation constant of the complex and the kinetics of its formation. The oxidized form of the coenzyme (DPN) did not form a spectrophotometrically identifiable complex with the protein. The molecular weight of the yeast enzyme is about twice that of the mammalian ADH and it is about 100 times more active. Neither DPN nor DPNH exhibits gross spectral shifts in the presence of the yeast enzyme. In order to study the coenzyme interactions of yeast ADH we have used the ultracentrifugal separation method which we had employed in previous studies with glyceraldehyde-3-phosphate dehydrogenases of rabbit muscle (6) and yeast (7). It was possible to detect complexes of yeast ADH with both DPN and DPNH and to measure their stoichiometry and apparent dissociation constants. The binding of DPN and DPNH by the yeast enzyme is competitive and the dissociation constants are in general agreement with the values derived from over-all reaction kinetics and competitive inhibition. There are still relatively few instances in which such correlations have been attempted and in two cases the reported Michaelis constant has been found to differ from the dissociation constant by more than two powers of 10 (5, 6). At high concentrations of yeast ADH the equilibrium of the oxidation reaction is shifted. The direction and magnitude of the shift may be accounted for by the formation of enzyme complexes with DPN, DPNH, and acetaldehyde.