Mechanism of Catalysis and Inhibition of Mammalian Protein Farnesyltransferase.

Abstract

Mammalian protein farnesyltransferase (FTase) catalyzes the transfer of a 15-carbon prenyl group from farnesyl diphosphate (FPP) to a cysteine residue near the carboxyl terminus of many proteins, including several key molecules involved in signal transduction. Common substrates include oncogenic Ras proteins, and several FTase inhibitors are under development for the treatment of various cancers. FTase is a member of the newest class of zinc metalloenzymes that catalyze sulfur alkylation, and the work described here provides further insight into the mechanism of catalysis for this enzyme, which may lead to an increased understanding of the substrate specificity and inhibition of FTase.

The reaction catalyzed by FTase results in two products: diphosphate and farnesylated protein or peptide. To measure the rate constant for diphosphate dissociation, a coupled fluorescent assay was developed. This assay can also be used to measure FTase activity for mechanistic studies and for high throughput screening to identify FTase substrates and inhibitors. The dissociation of the farnesylated product bound to FTase is accelerated by binding FPP. This step is crucial for substrate selectivity, as measured by substrate analog studies, and inhibition studies demonstrate that some FPP-competitive inhibitors function by slowing product dissociation. Together, these studies suggest that the binding of a second substrate molecule to facilitate product release is an important determinant of the substrate specificity, and potentially of the physiological regulation of FTase.

To investigate the structure of the chemical transition state of FTase, the primary 14C and α-secondary 3H kinetic isotope effects (KIEs) were measured using transient kinetics. These data suggest that the FTase reaction proceeds via a concerted mechanism with dissociative character, facilitated by the zinc ion which coordinates the thiolate of the peptide substrate. The effects of the Mg2+ concentration and mutations of positively charged residues that interact with the diphosphate leaving group on the α-secondary KIE suggest that Mg2+ and these side chains both stabilize the transition state for farnesylation and facilitate a conformational rearrangement of bound FPP that occurs prior to farnesylation. Finally, the dependence of the α-secondary KIE on peptide structure indicates that this FPP conformational change is important for substrate specificity.