STUDIES ON THE SUBSTRATE-BINDING SEGMENTS AND CATALYTIC ACTION OF LANOSTEROL SYNTHASE - AFFINITY LABELING WITH CARBOCATIONS DERIVED FROM MECHANISM-BASED ANALOGS OF 2,3-OXIDOSQUALENE AND SITE-DIRECTED MUTAGENESIS PROBES

Four 2,3-oxidosqualene analogs, 3, 4, 5, and 6, which are irreversible , time-dependent inhibitors of the enzyme lanosterol synthase, were fo und 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-1 8 reverse phase HPLC, and fragment identification by mass spectroscopy or Edman degradation. W232 and H234 are the targets of the chemical i nactivation by cations derived from analogs 3-6. 2,3-Oxidosqualene ana logs 7, 8, and 9 inactivated the enzyme with covalent attachment to th e 486-512 segment (Figure 3), which is in a domain that is predicted t o be an amphipathic alpha-helix. Site-directed mutagenesis of various amino acid residues (76 total) in lanosterol synthase which are conser ved in five different species has revealed that residues D456, H146, a nd H234 are essential for catalytic activity. These and other data per mit the formulation of a hypothetical working model of some aspects of the activation and binding of 2,3-oxidosqualene by lanosterol synthas e. The model is depicted in Figure 4. In that model D456 and protonate d H146 initiate cyclization, and the domains containing 231-236 and 48 6-512 make contact with the reacting substrate.