INFLUENCE OF METAL-IONS ON SUBSTRATE-BINDING AND CATALYTIC ACTIVITY OF MAMMALIAN PROTEIN GERANYLGERANYLTRANSFERASE TYPE-I

Protein geranylgeranyltransferase type-I (GGTase-I) transfers a gerany lgeranyl group from the prenyl donor geranylgeranyl diphosphate (GGPP) to the cysteine residue of substrate proteins containing a C-terminal CaaX-motif (a sequence motif of proteins consisting of an invariant C ys residue fourth from the C-terminus). The GGTase-I heterodimer conta ins one atom of zinc, and this metal is required for enzyme activity. In this regard, GGTase-I is similar to the related enzyme protein farn esyltransferase (FTase); the latter enzyme also requires Mg2+ for acti vity. The current studies were undertaken in an attempt to explore fur ther the role of bivalent metal ions in the activity of GGTase-I. Surp risingly, we found that GGTase-I and FTase have different metal requir ements. Specifically, in marked contrast to FTase, GGTase-I does not r equire Mg2+ for activity. Direct binding assays, including a novel flu orescence-based technique, were employed to obtain quantitative inform ation on the interaction of substrates with GGTase-I. Using these assa ys, we demonstrate that the Zn2+ in GGTase-I is required for peptide, but not for isoprenoid, substrate binding. Moreover, binding of GGPP p rotects GGTase-I from inactivation by zinc-chelating reagents; this pr otective effect is not seen with binding of peptide substrates. Metal substitution studies show that the Zn2+ in GGTase-I can be replaced by Cd2+, and that the Cd form of GGTase-I has altered specificity with r egard to utilization of both peptide and isoprenoid substrates. The si gnificance of these findings in relation to proposed mechanisms for th e GGTase-I reaction is discussed.