YEAST SQUALENE SYNTHASE - A MECHANISM FOR ADDITION OF SUBSTRATES AND ACTIVATION BY NADPH

Squalene synthase catalyzes the condensation of two molecules of farne syl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the s ubsequent reductive rearrangement of PSPP to squalene. Previous studie s of the mechanism of addition of FPP to the enzyme have led to confli cting interpretations of initial velocity measurements (Beytia, E., Qu reshi, A. A., and Porter, J. W. (1973) J. Biol. Chem. 248,1856-1867; A gnew, W. S., and Popjak, G. (1978) J. Biol. Chem. 253,4566-4573). Init ial velocities for synthesis of PSPP and squalene were measured over a wider range of FPP and NADPH concentrations than previously reported, using a soluble form of recombinant enzyme. In the absence of NADPH, PSPP formation was activated by FPP at concentrations above approximat ely 0.5 muM. At fixed levels of NADPH, the dependence of initial rates of PSPP and squalene synthesis on FPP concentrations indicated that t he C-15 substrate added by a sequential mechanism. In addition, NADPH stimulated synthesis of PSPP by 40-fold at saturating levels of the co factor. This stimulation is, at least in part, by reduction of PSPP to squalene.