Farnesyl protein transferase: Identification of K164 alpha and Y300 beta as catalytic residues by mutagenesis and kinetic studies

Farnesyl protein transferase (FPT) is an alpha/beta heterodimeric zinc enzy me that catalyzes posttranslational farnesylation of many key cellular regu latory proteins, including oncogenic Ras. On the basis of the recently repo rted crystal structure of FPT complexed with a CVIM peptide and alpha-hydro xyfarnesylphosphonic acid, site-directed mutagenesis of the FPT active site was performed so key residues that are responsible for substrate binding a nd catalysis could be identified. Eight single mutants, including K164N alp ha, Y166F alpha, Y166A alpha, Y200F alpha, H201A alpha, H248A beta, Y300F b eta, and Y361F beta, and a double mutant, H248A beta/Y300F beta, were prepa red. Steady-state kinetic analysis along with structural evidence indicated that residues Y200 alpha, H201 alpha, H248 beta, and Y361 beta are mainly involved in substrate binding, In addition, biochemical results confirm str uctural observations which show that residue Y166 alpha plays a key role in stabilizing the active site conformation of several FPT residues through c ation-pi interactions. Two mutants, K164N alpha and Y300F beta, have modera tely decreased catalytic constants (k(cat)). Pre-steady-state kinetic analy sis of these mutants from rapid quench experiments showed that the chemical step rate constant was reduced by 41- and 30-fold, respectively. The produ ct-releasing rate for each dropped approximately 10-fold, In pH-dependent k inetic studies, Y300F beta was observed to have both acidic and basic pK(a) values shifted 1 log unit from those of the wild-type enzyme, consistent w ith a possible role for Y300 beta as an acid-base catalyst. K164N alpha had a pK(a) shift from 6.0 to 5,3, which suggests it may function as a general acid. On the basis of these results along with structural evidence, a poss ible FPT reaction mechanism is proposed with both Y300 beta and K164 alpha playing key catalytic roles in enhancing the reactivity of the farnesyl dip hosphate leaving group.