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 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.
Hawes
J W
JW
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122, USA.
Crabb
D W
DW
Chan
R M
RM
Rougraff
P M
PM
Harris
R A
RA
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry
D011994:Recombinant Proteins; D009243:NAD; D014443:Tyrosine; D000429:Alcohol Oxidoreductases; C021398:3-hydroxyisobutyrate dehydrogenase; D013774:Tetranitromethane; D003545:Cysteine