Divalent cation and prenyl pyrophosphate specificities of the protein farnesyltransferase from rat brain, a zinc metalloenzyme

The separate catalytic roles of Zn²⁺ and Mg²⁺ and the specificity of the prenyl pyrophosphate-binding site of the rat brain protein farnesyltransferase were explored using a purified enzyme preparation. The binding of p21^Hras to the enzyme was abolished by dialysis against EDTA and restored by addition of ZnCl₂, as demonstrated by chemical cross-linking. The binding of the other substrate, farnesyl pyrophosphate, was independent of divalent cations, as demonstrated by gel filtration. Transfer of the enzyme-bound farnesyl group to the bound p21^Hras required Mg²⁺. Geranylgeranyl pyrophosphate bound to the prenyl pyrophosphate-binding site with an affinity equal to that of farnesyl pyrophosphate, but the geranylgeranyl group was not transferred efficiently to p21^Hras. It also was not transferred to a modified p21^Hras containing COOH-terminal leucine, a protein that was shown previously to be a good substrate for a rat brain geranylgeranyltransferase. We conclude that the protein farnesyltransferase is a metalloenzyme that most likely contains Zn²⁺ at the peptide-binding site. It thus resembles certain metallopeptidases, including carboxypeptidase A and the angiotensin-converting enzyme. Strategies previously developed to screen for inhibitors of those enzymes may aid in the search for inhibitors of the protein farnesyltransferase.