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 microM 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.
Marshall
J A
JA
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409, USA.
Nes
W D
WD
D000595:Amino Acid Sequence; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry* / Q000302:isolation & purification*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013997:Time Factors
D010455:Peptides; D008780:Methyltransferases