Substitution of arginine for histidine-47 in the coenzyme binding site of yeast alcohol dehydrogenase I.
Molecular modeling of alcohol dehydrogenase suggests that His-47 in the yeast enzyme (His-44 in the protein sequence, corresponding to Arg-47 in the horse liver enzyme) binds the pyrophosphate of the NAD coenzyme. His-47 in the Saccharomyces cerevisiae isoenzyme I was substituted with an arginine by a directed mutation. Steady-state kinetic results at pH 7.3 and 30 degrees C of the mutant and wild-type enzymes were consistent with an ordered Bi-Bi mechanism. The substitution decreased dissociation constants by 4-fold for NAD+ and 2-fold for NADH while turnover numbers were decreased by 4-fold for ethanol oxidation and 6-fold for acetaldehyde reduction. The magnitudes of these effects are smaller than those found for the same mutation in the human liver beta enzyme, suggesting that other amino acid residues in the active site modulate the effects of the substitution. The pH dependencies of dissociation constants and other kinetic constants were similar in the two yeast enzymes. Thus, it appears that His-47 is not solely responsible for a pK value near 7 that controls activity and coenzyme binding rates in the wild-type enzyme. The small substrate deuterium isotope effect above pH 7 and the single exponential phase of NADH production during the transient oxidation of ethanol by the Arg-47 enzyme suggest that the mutation makes an isomerization of the enzyme-NAD+ complex limiting for turnover with ethanol.
Department of Biochemistry, University of Iowa, Iowa City 52242.
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RM
Gould
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Plapp
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000235:genetics; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004271:DNA, Fungal; D009243:NAD; D006639:Histidine; D001120:Arginine; D000426:Alcohol Dehydrogenase