Yeast sterol C24-methyltransferase: role of highly conserved tyrosine-81 in catalytic competence studied by site-directed mutagenesis and thermodynamic analysis.

摘要: Abstract The role of Try-81 in the reaction catalyzed by Saccharomyces cerevisiae sterol 24-C-methyltransferase (Erg6p) was investigated kinetically and for product differences against a panel position-81 mutants which Tyr substituted with Trp, Phe, Ile, Leu, Val Ala. residue chosen mutation is one that reported previously to accept fecosterol yield set 24-ethyl (idene) products typical plants, showing amino acid located close transient C25 carbocation intermediate active site. One group (aromatic) tested natural substrate zymosterol accelerated C-methylation ( k cat / K m ) whereas other (aliphatics) decreased catalytic competence as side chain downsized. Mutating aromatic assaying analog designed suicide 26,27-dehydrozymosterol favored C26-monol formation, mutating aliphatic smaller size C26-diol formation (a measure enzyme alkylation). In no case converted an formed C25-diol. Thermodynamic analysis (determination E , Δ G ‡ H T S performed these enzymes assayed its or paired “charged’ high energy (HEI) analogs 24( R,S )25,epiminolanosterol 25-azalanosterol “neutral” membrane insert ergosterol showed aromatics can reduce inhibitor potency (measured i ), yet catalysis improve Trp81 introduction gain free associated stabilization transition state rate-controlling step directed toward turnover. Alternatively, chains led destabilization site structure accompanied increases partition ratios abortive complex formation. results are explained consideration functional attributed Tyr81 substitution aliphatics different involved cation-π hydrogen bonding interactions activation barriers required differing conformations orient reactants direction turnover versus inactivation.