The structure and kinetics of the
isozymes from Saccharomyces cerevisiae (ADH I, II, III) have been compared, and the ADH I gene was specifically mutagenized in order to substitute
amino acid residues of particular interest. A model of the yeast
enzyme was constructed on the basis of the structure of the homologous horse liver
enzyme. Steady state kinetic studies, at pH 7.3 and 30 degrees C, showed that the
enzymes follow the Ordered Bi Bi mechanism. ADH II has a Michaelis constant for
ethanol that is 10-fold smaller than the constants found for ADH I or III. Replacement of Met-294 (liver numbering) in the substrate binding pocket of ADH I with Leu, as found in ADH II, could be responsible for the different kinetics. However, the mutant
enzyme, ADH I-Leu, had constants with
ethanol that were similar to those of ADH I. Nevertheless, the Leu
enzyme had better catalytic activity with longer chain
alcohols than did the Met
enzyme. Other substitutions must account for the differences between ADH I and II. His-47 binds the
pyrophosphate of
coenzyme, and replacement with Arg (as in the liver
enzyme) decreases turnover numbers by 6-fold and dissociation constants for NAD+ and
NADH by only 2 to 4-fold. The lower turnover number explains why yeast harboring the mutant Arg
enzyme are resistant to
poisoning by
allyl alcohol. ADH I and ADH I-Arg have maximal activity on
ethanol at pH values above a pK of about 7. Replacement of His-51 with Gln reduces activity 12-fold and abolishes the pK value.(ABSTRACT TRUNCATED AT 250 WORDS)