Mechanistic studies of rat protein farnesyltransferase indicate an associative transition state

Protein farnesyltransferase is a zinc metalloenzyme that catalyzes the tran sfer of a 15-carbon farnesyl group to a conserved cysteine residue of a pro tein substrate. Both electrophilic and nucleophilic mechanisms have been pr oposed for this enzyme. In this work, we investigate the detailed catalytic mechanism of mammalian protein farnesyltransferase by measuring the effect of metal substitution and/or substrate alterations on the rate constant of the chemical step. Substitution of cadmium for the active site zinc enhanc es peptide affinity approximately 5-fold and decreases the rate constant fo r the formation of the thioether product approximately 6-fold, indicating c hanges in the metal-thiolate coordination in the catalytic transition state . In addition, the observed rate constant for product formation decreases f or C3 fluoromethyl farnesyl pyrophosphate substrates, paralleling the numbe r of fluorines at the C3 methyl position and indicating that a rate-contrib uting transition state has carbocation character. Magnesium ions do not aff ect the affinity of either the peptide or the isoprenoid substrate but spec ifically enhance the observed rate constant for product formation 700-fold, suggesting that magnesium coordinates and activates the diphosphate leavin g group. These data suggest that FTase catalyzes protein farnesylation by a n associative mechanism with an "exploded" transition state where the metal -bound peptide/protein sulfur has a partial negative charge, the C1 of FPP has a partial positive charge, and the bridge oxygen between C1 and the cr phosphate of FPP has a partial negative charge. This proposed transition st ate suggests that stabilization of the developing charge on the carbocation and pyrophosphate oxygens is an important catalytic feature.