Role of the three cysteines in the catalysis and stability of ketosteroid isomerase from pseudomonas putida biotype b

Suhng Wook Kim, Soyoung Ju, Gildon Choi, Byung Ha Oh, Kwan Yong Choi

Research output: Contribution to journalArticle

Abstract

As-3-ketosteroid isomerase (KSI) catalyzes the conversion of a variety of As 3-ketosteroids to their conjugated A4-3-ketosteroids. Although many studies of the mechanism of the KSI have identified tyrosine and aspartate as the catalyticaily essential general acid and base, the existence of an as yet unidentified functional group in the KSI active site has been suggested. Previous active sitedirected photoaffinity labeling study of KSI from Pseudomonas putida biotype B showed that one of the cysteine residues is susceptible to catalytic modification. In order to identify the catalytic roles of cysteine residues in P. putida KSI, each of the three cysteinyl residues per subunit has been changed to a serine residue by site-directed mutagenesis. Mutations of each of the three cysteine residues lowered k at values only slightly to 83% - 99% compared with the wild-type enzyme. None of the matations of the three cysteines severely destabilized the protein. In the native conformation, Cys 69 appears to have a exposed sulfhydryl group, Cys 97 have a semi-buried sulfhydryl group, and Cys 97 have a fully buried sulfhydryl group as reflected by their reactivity toward 5,5'-dithiobis-(2-nitrobenzoate) (DTNB). However, modification of the exposed sulfhydryl group with DTNB has almost no effect on enzyme activity. The results demonstrate that none of the cysteines of KSI from P. putida is essential for catalytic activity or stability. Preliminary three-dimensional crystal structure of P. putida KSI also supports these results.

Original languageEnglish
JournalFASEB Journal
Volume11
Issue number9
Publication statusPublished - 1997 Dec 1
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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