The catalytic utilization of dimethylallyl, geranyl, farnesyl, and geranylgeranyl diphosphates in the reaction catalyzed by recombinant human farnesyl:protein transferase (hFPTase) has been examined in the presence of three different protein substrates, Ras-CVLS, Ras-CVIM, and Ras-CAIL. hFPTase catalyzed both farnesylation and geranylation of Ras-CVLS and of Ras-CVIM but not of Ras-CAIL. Geranylgeranylation was observed, but only when Ras-CVIM was the acceptor substrate. Steady-state initial velocity and dead-end inhibitor studies indicate that hFPTase-catalyzed geranylation, like bovine FPTase-catalyzed farnesylation, proceeds through a random order, sequential mechanism. Surprisingly, however, Michaelis constants for a given protein acceptor substrate varied depending upon which isoprenoid diphosphate was used as the donor substrate, showing that these substrates do not bind independently to the enzyme (under catalytic conditions). In addition, at very high concentrations of Ras-CVIM, substrate inhibition was observed in the presence of both FPP and GPP. Isotope partitioning studies showed that, at high concentrations of Ras-CVIM, more than 80% of the bound farnesyl diphosphate (FPP) can be trapped as product, suggesting that the binary complex is catalytically competent and that the ternary, complex proceeds to product faster than it releases FPP. The release rate of FPP from the binary complex was calculated to be 0.05 s-1, which is only about eight times greater than k(cat). Thus, the binding of FPP to the enzyme in the presence of the protein substrate is not an equilibrium situation. The isotope partitioning studies together with the steady-state kinetic data lead us to conclude that the mechanism is random in principle (since both substrates appear to bind independently to free enzyme) but ordered in practice, since the preferred catalytic pathway is through the FPTase.FPP binary complex. Computer simulations of a steady-state, random order sequential mechanism using experimentally determined kinetic constants as constraints provide a model which describes the observed behavior.
