Mechanism and structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase from Saccharomyces cerevisiae.
The mechanism of action and active site of the enzyme (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase (SMT) from Saccharomyces cerevisiae strain GL7 have been probed with AdoMet, (S)-adenosyl-L-homocysteine, a series of 35 sterol substrates as acceptor molecules and a series of 10 substrate and high energy intermediate (HEI) sterol analogues as inhibitors of biomethylation. The SMT was found to be selective for sterol, both regio- and stereochemically. The presence of an unhindered 24,25-bond, an equatorially-oriented polar group at C-3 (which must act as a proton acceptor) attached to a planar nucleus and a freely rotating side chain were obligatory structural features for sterol binding/catalysis; no essential requirement or significant harmful effects could be found for the introduction of an 8(9)-bond, 14 alpha-methyl or 9 beta,19-cyclopropyl group. Alternatively, methyl groups at C-4 prevented productive sterol binding to the SMT. Initial velocity, product inhibition, and dead-end experiments demonstrated a rapid-equilibrium random bi bi mechanism. Deuterium isotope effects developed from SMT assays containing mixtures of [3-3H]zymosterol with AdoMet or [methyl-2H3]AdoMet confirmed the operation of a random mechanism, kappa H/kappa D = 1.3. From this combination of results, the spatial relationship of the sterol substrate to AdoMet could be approximated and the topology of the sterol binding to the SMT thereby formulated.
Venkatramesh
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Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, 79409-1061 USA.
Guo
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Jia
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D002458:Cell Fractionation; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D008954:Models, Biological; D012436:S-Adenosylmethionine / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012995:Solubility; D013261:Sterols / Q000378:metabolism*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D013261:Sterols; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase