Protein farnesyltransferase (FTase) is a zinc metalloenzyme that catalyzes the prenylation of several proteins that are important in cellular regulatory events. A specific residue of FTase, Cys299 in the â subunit previously identified as essential for zinc binding and catalysis, had been tentatively assigned as one of the zinc ligands. This assignment was subsequently confirmed in the X-ray structure of FTase, which also identified two additional residues, Asp297 and His362 in the â subunit, as the remaining protein-derived metal ligands. To more fully explore the role of zinc in the catalytic mechanism of FTase, site-directed mutagenesis was performed on these two zinc ligands. Although the abilities of all the mutants to bind the farnesyl diphosphate substrate were similar to that of the wild-type enzyme, all the mutants displayed markedly reduced enzymatic activities and zinc affinities. Steady-state and pre-steadystate kinetic analyses of the residual activities indicated that the rate-limiting step changed from product release in the wild-type enzyme to the chemical step of product formation for three of the mutant enzymes. Additionally, single-turnover experiments indicated that the greatest effect of alteration of zinc ligands for all the mutants was on the product formation step, this being reduced 10^3-10^5-fold in the mutant forms compared to the wild-type enzyme. These results confirm a critical involvement of the zinc in catalysis by FTase and support a model in which the metal ion is directly involved in the chemical step of the enzymatic reaction.