Methodology for the Preparation of Pure Recombinant S. cerevisiae Lanosterol Synthase Using a Baculovirus Expression System. Evidence That Oxirane Cleavage and A-Ring Formation Are Concerted in
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- 作者：E. J. Corey ; Hengmiao Cheng ; C. Hunter Baker ; Seiichi P. T. Matsuda ; Ding Li ; and Xuelei Song
- 刊名：Journal of the American Chemical Society
- 出版年：1997
- 出版时间：February 12, 1997
- 年：1997
- 卷：119
- 期：6
- 页码：1277 - 1288
- 全文大小：392K
- 年卷期：v.119,no.6(February 12, 1997)
- ISSN：1520-5126
文摘
Lanosterol synthase [(S)-2,3-epoxysqualene mutase(cyclizing, lanosterol forming), EC 5.4.99.7], the enzymefrom Saccharomyces cerevisiae which catalyzes the complexcyclization/rearrangement step in sterol biosynthesis,was overexpressed in baculovirus-infected cells and purified tohomogeneity in three steps. Using pure enzyme thekinetics of cyclization were determined using Michaelis-Mentenanalysis for 2,3-oxidosqualene (1) and twoanalogsin which the C-6 methyl was replaced by H (3) or Cl(4). The measuredVmax/KM ratios for1, 3, and 4 were foundto be 138, 9.4, and 21.9, respectively, a clear indication that oxiranecleavage and cyclization to form the A-ring areconcerted, since the nucleophilicity of the proximate double bondinfluences the rate of oxirane cleavage. No catalyticmetal ions could be detected in purified lanosterol synthase by atomicabsorption analysis. Site-directed mutagenesisstudies of each of the six strongly conserved aspartic acid residues (D
N mutation) and each of the nine conservedglutamic acid residues (E Q) revealed that only one, D456, isessential for catalytic function of the enzyme. Theessential D456 residue is a likely candidate for electrophilic(specifically protic) activation of the oxirane function.
N mutation) and each of the nine conservedglutamic acid residues (E Q) revealed that only one, D456, isessential for catalytic function of the enzyme. Theessential D456 residue is a likely candidate for electrophilic(specifically protic) activation of the oxirane function.