Positively charged side chains in protein farnesyltransferase enhance catalysis by stabilizing the formation of the diphosphate leaving group

Protein farnesyltransferase (FTase) requires both Zn2+ and Mg2+ for efficient catalysis of the formation of a thioether bond between carbon-1 of farnesyldiphosphate (FPP) and the cysteine thiolate contained in the carboxy-terminal CaaX sequence of target proteins. Millimolar concentrations of Mg2+ accelerate catalysis by as much as 700-fold in FTase. Although FTase lacks a typical DDXXD Mg2+ binding site found in other enzymes that use Mg2+ for diphosphate stabilization, D352beta in FTase has been implicated in binding Mg2+ (Pickett et al. (2003) J. Biol. Chem. 278, 51243). Structural studies demonstrate that the diphosphate (PPi) group of FPP resides in a binding pocket made up of highly positively charged side chains, including residues R291beta and K294beta, prior to formation of an active conformation. Analysis of the Mg2+ dependence of FTase mutants demonstrates that these positively charged residues decrease the Mg2+ affinity up to 40-fold. In addition, these residues enhance the farnesylation rate constant by almost 80-fold in the presence of Mg2+, indicating that these residues are not simply displaced by Mg2+ during the reaction. Mutations at R291beta increase the pK(a) observed in the magnesium affinity, suggesting that this arginine stabilizes the deprotonated form of the PPi leaving group. Furthermore, binding and catalysis data using farnesylmonophosphate (FMP) as a substrate indicate that the side chains of R291beta and K294beta interact mainly with the beta-phosphate of FPP during the chemical reaction. These results allow refinement of the model of the Mg2+ binding site and demonstrate that positive charge stabilizes the developing charge on the diphosphate leaving group.