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

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 Ki of 1.1 μM and kinact 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 ( , stretching between T78 and F91) characteristic of SMT enzymes. The results confirm that is the sterol binding/active site.