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 3). The attachment was determined by tryptic digestion of the inactivated enzyme, separation of the tryptic cleavage products by C-18 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 3), which is in a domain that is predicted to be an amphipathic alpha-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 4. In that model D456 and protonated H146 initiate cyclization, and the domains containing 231-236 and 486-512 make contact with the reacting substrate.