Farnesyl diphosphate synthase. Altering the catalytic site to select for geranyl diphosphate activity

Farnesyl diphosphate synthase (FPPase) catalyzes chain elongation of the C- 5 substrate dimethylallyl diphosphate (DMAPP) to the C-15 product farnesyl diphosphate (FPP) by addition of two molecules of isopentenyl diphosphate ( IPP). The synthesis of FPP proceeds in two steps, where the C-10 product of the first addition, geranyl diphosphate (GPP), is the substrate for the se cond addition. The product selectivity of avian FPPase was altered to favor synthesis of GPP by site-directed mutagenesis of residues that form the bi nding pocket for the hydrocarbon residue of the allylic substrate. Amino ac id substitutions that reduced the size of the binding pocket were identifie d by molecular modeling. FPPase mutants containing seven promising modifica tions were constructed. Initial screens using DMAPP and GPP as substrates i ndicated that two of the substitutions, A116W and N144'W, strongly discrimi nated against binding of GPP to the allylic site. These observations were c onfirmed by an analysis of the products from reactions with DMAPP in the pr esence of excess IPP and by comparing the steady-state kinetic constants fo r the wild-type enzyme and the A116W and N114W mutants.