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J Org Chem. 1999 Mar 5;64(5):1535-1542.

Site-Directed Mutagenesis of the Sterol Methyl Transferase Active Site from Saccharomyces cerevisiae Results in Formation of Novel 24-Ethyl Sterols.

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Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409.


Delta(24(28))-Sterols are end products of a mono C-methylation pathway catalyzed by the native Delta(24(25))- to Delta(24(28))-sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae. Using a Tyr(81) to Phe mutant SMT enzyme of S. cerevisiae, generated by site-directed mutagenesis of a highly conserved residue in the sterol binding site, we found that several Delta(24(25))- and Delta(24(28))-sterols, which are not substrates for the native protein, were catalyzed to mono- and bis-C24-alkylated side chains. The mutant protein behaved similarly to the native protein in chromatography and in binding zymosterol, the preferred substrate. Zymosterol was converted to fecosterol by the Y81F mutant protein with similar turnover efficiency as the native protein (K(m) = 12 &mgr;M and k(cat) = 0.01 s(-)(1)); trace 24-ethyl sterols were detected from these incubations. 4alpha-Methyl zymosterol, which is not a normal substrate for the wild-type SMT enzyme, was converted to 4alpha-methyl fecosterol in high yield. When fecosterol and 4alpha-methyl fecosterol were assayed individually at saturating concentrations only fecosterol served as an effective substrate for the second C-transfer step (K(m) = 38 &mgr;M and k(cat) = 0.002 s(-)(1)), suggesting that successive C-methylation of Delta(24(28))-substrates is limited by product release and that molecular recognition of sterol features involves hydrogen bond formation. Isomeric 24-ethyl sterol olefins generated from 24(28)-methylene cholesterol were characterized by chromatographic (GC and HPLC) and spectral methods (MS and (1)H NMR), viz., fucosterol, isofucosterol, and clerosterol. Changes in rate of C-methylation and product distributions resulting from deuterium substitution at C28 were used to establish the kinetic isotope effects (KIEs) for the various deprotonations leading to C24-methylene, C24-ethylidene, and C24-ethyl sterols. An isotope effect on C28 methyl deprotonation generated during the first C(1)-transfer was detected with zymosterol and desmosterol paired with AdoMet and [(2)H(3)-methyl]AdoMet. A similar experiment to test for a KIE generated during the second C(1)-transfer reaction with AdoMet paired with 24(28)-methylenecholesterol and [28-(2)H(2)]24(28)-methylene cholesterol indicated an inverse isotope effect associated with C27 deprotonation. Alteration in the proportion of the C24 alkylated olefinic products generated by the pure Y81F mutant resulted from the suppression of the formation of Delta(24(28))-ethylidene sterols (C28 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of 24-ethyl sterols (C27 deprotonation). Kinetic study on the rate of product formation indicated a normal KIE of k(H)/k(D) = 2.62 for the first C(1)-transfer. Alternatively, an inverse KIE was established with k(H)/k(D) = 0.9 for the second C(1)-transfer resulting from conversion of the 24(28)-double bond (sp(2) hybridization) to a 24beta-ethyl group (sp(3) hybridization). From the structures and stereochemical assignments of the C-ethyl olefin products, the stereochemistry of the attack of AdoMet in the second C(1)-transfer was found to operate a Si-face (backside) attack at C24, analogous to the first C(1)-transfer reaction.

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