Chemical Modification and Site-Directed Mutagenesis Studies of Rat 3-Hydroxyisobutyrate Dehydrogenase

John W. Hawes, David Crabb, Rebecca Chan, Robert Harris, Paul M. Rougraff

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 Vmaxthan wild-type enzyme, but Kmvalues 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.

Original languageEnglish (US)
Pages (from-to)4231-4237
Number of pages7
JournalBiochemistry
Volume34
Issue number13
DOIs
StatePublished - Sep 1 1995

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3-hydroxyisobutyrate dehydrogenase
Mutagenesis
Chemical modification
Site-Directed Mutagenesis
Rats
Enzymes
Cysteine
Alcohol Dehydrogenase
Phenylmethylsulfonyl Fluoride
Iodoacetates
Catalysis
Sequence Homology

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chemical Modification and Site-Directed Mutagenesis Studies of Rat 3-Hydroxyisobutyrate Dehydrogenase. / Hawes, John W.; Crabb, David; Chan, Rebecca; Harris, Robert; Rougraff, Paul M.

In: Biochemistry, Vol. 34, No. 13, 01.09.1995, p. 4231-4237.

Research output: Contribution to journalArticle

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