CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS STUDIES OF RAT 3-HYDROXYISOBUTYRATE DEHYDROGENASE

Rat 3-hydroxyisobutyrate dehydrogenase shares sequence homology with the short-chain alcohol dehydrogenases. Site-directed mutagenesis and chemical modifications were used to examine the roles of cysteine residues and other residues conserved in this family of enzymes. It was found that a highly conserved tyrosine residue, Y162 in S-hydroxyisobutyrate dehydrogenase, does not function catalytically as it may in other short-chain alcohol dehydrogenases. Of the six cysteine residues present in 3-hydroxyisobutyrate dehydrogenase, only cysteine 215 was found to be critical to catalysis. C215A and C215D mutant enzymes were catalytically inactive but produced CD spectra identical to wild-type enzyme. C215S mutant enzyme displayed a lowered V-max than wild-type enzyme, but K-m values were similar to those of wild-type enzyme. The C215S mutant enzyme was inactivated by treatment with phenylmethanesulfonyl fluoride but was not inactivated by treatment with iodoacetate, whereas the wild-type enzyme was inactivated by treatment with iodoacetate but not inactivated by treatment with phenylmethanesulfonyl fluoride. The present data suggest that 3-hydroxyisobutyrate dehydrogenase differs in mechanism from other shea-chain alcohol dehydrogenases studied to date and that cysteine 215 has a critical function in catalysis, possibly as a general base catalyst.