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  • Title: Chemical modification and site-directed mutagenesis studies of rat 3-hydroxyisobutyrate dehydrogenase.
    Author: Hawes JW, Crabb DW, Chan RM, Rougraff PM, Harris RA.
    Journal: Biochemistry; 1995 Apr 04; 34(13):4231-7. PubMed ID: 7703236.
    Abstract:
    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 3-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 Vmax than wild-type enzyme, but Km 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 short-chain alcohol dehydrogenases studied to date and that cysteine 215 has a critical function in catalysis, possibly as a general base catalyst.
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