Squalene synthase: Steady-state, pre-steady-state, and isotope-trapping studies

Squalene synthase catalyzes two consecutive reactions in sterol biosynthesi s the condensation of two molecules of farnesyl diphosphate (FPP) to form t he cyclopropylcarbinyl intermediate presqualene diphosphate (PSPP) and the subsequent rearrangement and reduction of PSPP to form squalene. Steady-sta te and pre-steady-state kinetic studies, in combination with isotope-trappi ng experiments of enzyme substrate complexes, indicate that two molecules o f FPP add to the enzyme before NADPH: and that PSPP is converted directly t o squalene without dissociating from the enzyme under normal catalytic cond itions. In addition, formation of PSPP or a prior conformational change in squalene synthase is the rate-limiting step for synthesis of squalene from FPP via PSPP in the presence of NADPH and for synthesis of PSPP in the abse nce of NADPH. Squalene synthase is inhibited at high concentrations of FPP. Inhibition is specific for the formation of squalene, but not PSPP, and is competitive with respect to NADPH. In addition, the binding of either NADP H or a third, nonreacting molecule of FPP stimulates the rate of PSPP forma tion. A kinetic mechanism is proposed to account for these observations.