Structure and function in yeast alcohol dehydrogenases.
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)
Plapp
B V
BV
Ganzhorn
A J
AJ
Gould
R M
RM
Green
D W
DW
Hershey
A D
AD
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D001120:Arginine; D001665:Binding Sites; D006639:Histidine; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D007527:Isoenzymes; D006639:Histidine; D001120:Arginine; D000426:Alcohol Dehydrogenase; D007930:Leucine