Yeast sterol C24-methyltransferase: role of highly conserved tyrosine-81 in catalytic competence studied by site-directed mutagenesis and thermodynamic analysis.
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 of position-81 mutants in which Tyr was substituted with Trp, Phe, Ile, Leu, Val and Ala. The residue chosen for mutation is one that was reported previously to accept fecosterol and yield a set 24-ethyl (idene) sterol products typical of plants, showing the amino acid residue is located close to the transient C25 carbocation intermediate in the active site. One group of mutants (aromatic) tested with the natural substrate zymosterol accelerated the C-methylation reaction (k(cat)/K(m)) whereas the other group of mutants (aliphatics) decreased catalytic competence as the amino acid side chain was downsized. Mutating to aromatic and assaying with the substrate analog designed as a suicide substrate 26,27-dehydrozymosterol favored C26-monol formation, whereas mutating to the aliphatic of smaller size favored C26-diol formation (a measure of enzyme alkylation). In no case was zymosterol converted to an intermediate that formed a C25-diol. Thermodynamic analysis (determination of E(a), DeltaG(double dagger), DeltaH(double dagger) and TDeltaS(double dagger)) for the C-methylation reaction performed by these enzymes assayed with the substrate and its analog or zymosterol paired with the "charged' high energy intermediate (HEI) analogs 24(R,S)25,epiminolanosterol and 25-azalanosterol or "neutral" membrane insert ergosterol showed that mutation to aromatics can reduce inhibitor potency (measured as K(m)/K(i)), yet catalysis can improve in Trp81 by the introduction of a gain in free energy associated with stabilization of the transition state of a rate-controlling step directed toward turnover. Alternatively, mutation to the smaller aliphatic amino acid side chains led to a destabilization in the active site structure which was accompanied by increases in the partition ratios associated with abortive complex formation. The results are explained by consideration of the functional differences attributed to Tyr81 substitution to aromatics and aliphatics of different size involved with cation-pi or hydrogen bonding interactions and in the activation barriers required of differing side chain conformations to orient the reactants in the direction of turnover versus enzyme inactivation.
Department of Chemistry and Biochemistry, Texas Tech University, PO Box 1061, Lubbock, TX 79409-1061, USA. wdavid.nes@ttu.edu
W David
WD
Nes
Pruthvi
P
Jayasimha
Zhihong
Z
Song
D002384:Catalysis; D003198:Computer Simulation; D017124:Conserved Sequence; D004789:Enzyme Activation; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry*
D014443:Tyrosine; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase