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 measured
Vmax/
KM ratios for
1,
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
![](/images/entities/rarr.gif)
N mutation) and each of the nine conservedglutamic acid residues (E
![](/images/entities/rarr.gif)
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.