Lanosterol Synthase - Mechanism

Mechanism

Though some data on the mechanism has been obtained by the use of suicide inhibitors, mutagenesis studies, and homology modeling, it is still not fully understood how the enzyme catalyzes the formation of lanosterol.

Initial Epoxide Protonation and Ring Opening: Before the acquisition of the protein’s X-ray crystal structure, site-directed mutagenesis was used to determine residues key to the enzyme’s catalytic activity. It was determined that an aspartic acid residue (D455) and two histidine residues (H146 and H234) were essential to enzyme function. Corey et al. hypothesized that the aspartic acid acts by protonating the substrate’s epoxide ring, thus increasing its susceptibility to intramolecular attack by the nearest double bond, with H146 possibly intensifying the proton donor ability of the aspartic acid through hydrogen bonding. After acquisition of the X-ray crystal structure of the enzyme, the role of D455 as a proton donor to the substrate’s epoxide was confirmed, though it was found that D455 is more likely stabilized by hydrogen bonding from two cysteine residues (C456 and C533) than from the earlier suggested histidine.

Ring Formation Cascade: Epoxide protonation activates the substrate, setting off a cascade of ring forming reactions. Four rings in total (A through D) are formed, producing the cholesterol backbone. Though the idea of a concerted formation of all four rings had been entertained in the past, kinetic studies with (S)-2,3-oxidosqualene analogs showed that product formation is achieved through discrete carbocation intermediates (see Figure 1). Isolation of monocyclic and bicyclic products from lanosterol synthase mutants has further weakened the hypothesis of a concerted mechanism. Evidence suggests that epoxide ring opening and A ring formation is concerted, though.