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Four 2,3-oxidosqualene analogs, 3, 4, 5, and 6, which are irreversible, time-dependent inhibitors of the enzyme lanosterol synthase, were found to attach covalently within the 231−236 (yeast numbering) segment (Figure ). The attachment was determined by tryptic digestion of the inactivated enzyme, separation of the tryptic cleavage products by C18 reverse phase HPLC, and fragment identification by mass spectroscopy or Edman degradation. W232 and H234 are the targets of the chemical inactivation by cations derived from analogs 3−6. 2,3-Oxidosqualene analogs 7, 8, and 9 inactivated the enzyme with covalent attachment to the 486−512 segment (Figure ), which is in a domain that is predicted to be an amphipathic α-helix. Site-directed mutagenesis of various amino acid residues (76 total) in lanosterol synthase which are conserved in five different species has revealed that residues D456, H146, and H234 are essential for catalytic activity. These and other data permit the formulation of a hypothetical working model of some aspects of the activation and binding of 2,3-oxidosqualene by lanosterol synthase. The model is depicted in Figure . In that model D456 and protonated H146 initiate cyclization, and the domains containing 231−236 and 486−512 make contact with the reacting substrate.

Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme from Saccharomyces cerevisiae which catalyzes the complex cyclization/rearrangement step in sterol biosynthesis, was overexpressed in baculovirus-infected cells and purified to homogeneity in three steps. Using pure enzyme the kinetics of cyclization were determined using Michaelis−Menten analysis for 2,3-oxidosqualene (1) and two analogs in which the C−6 methyl was replaced by H (3) or Cl (4). The measured V max/K M ratios for 1, 3, and 4 were found to be 138, 9.4, and 21.9, respectively, a clear indication that oxirane cleavage and cyclization to form the A-ring are concerted, since the nucleophilicity of the proximate double bond influences the rate of oxirane cleavage. No catalytic metal ions could be detected in purified lanosterol synthase by atomic absorption analysis. Site-directed mutagenesis studies of each of the six strongly conserved aspartic acid residues (D → N mutation) and each of the nine conserved glutamic acid residues (E → Q) revealed that only one, D456, is essential for catalytic function of the enzyme. The essential D456 residue is a likely candidate for electrophilic (specifically protic) activation of the oxirane function.

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