Isolation and characterization of an active-site peptide from a sterol methyl transferase with a mechanism-based inhibitor

Julie A. Marshall, W. David Nes

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Chemical affinity labeling of pure sterol methyl transferase (SMT) from Saccharomyces cerevisiae using the mechanism-based irreversible inhibitor, [3-3H]26,27-dehydrozymosterol, inhibited the SMT with an apparent K(i) of 1.1 μM and k(inact) of 1.52 min-1. The protein-inhibitor adduct was subjected to cleavage with trypsin and the resulting covalently modified peptide was analyzed by Edman sequencing from the N-terminus. The radiochemically labeled ca. 5.0 kDa peptide fragment of the cleavage mixture was shown to be contiguous through 17 residues to a segment that includes a highly conserved hydrophobic motif (Region I. stretching between T78 and F91) characteristic of SMT enzymes. The results confirm that Region I is the sterol binding/active site.

Original languageEnglish
Pages (from-to)1533-1536
Number of pages4
JournalBioorganic and Medicinal Chemistry Letters
Volume9
Issue number11
DOIs
StatePublished - Jun 7 1999

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