Influence of metal ions on substrate binding and catalytic activity of mammalian protein geranylgeranyltransferase type-I

Abstract

Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl 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 Cys residue fourth from the C-terminus). The GGTase-I heterodimer contains one atom of zinc, and this metal is required for enzyme activity. In this regard, GGTase-I is similar to the related enzyme protein farnesyltransferase (FTase); the latter enzyme also requires Mg2+ for activity. The current studies were undertaken in an attempt to explore further the role of bivalent metal ions in the activity of GGTase-I. Surprisingly, we found that GGTase-I and FTase have different metal requirements. Specifically, in marked contrast to FTase, GGTase-I does not require Mg2+ for activity. Direct binding assays, including a novel fluorescence-based technique, were employed to obtain quantitative information on the interaction of substrates with GGTase-I. Using these assays, we demonstrate that the Zn2+ in GGTase-I is required for peptide, but not for isoprenoid, substrate binding. Moreover, binding of GGPP protects GGTase-I from inactivation by zinc-chelating reagents; this protective 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 regard to utilization of both peptide and isoprenoid substrates. The significance of these findings in relation to proposed mechanisms for the GGTase-I reaction is discussed.

Lysine β311 of Protein Geranylgeranyltransferase Type I Partially Replaces Magnesium

“…Previous kinetic data suggest that the kinetic scheme for GGTase I is similar to that of FTase (34,35), including that the substrate binding is functionally ordered with GGPP binding before peptide and that the rate constant for geranylgeranylation is faster than product dissociation (Scheme 1). The kinetic and thermodynamic data that we have determined for GGTase I are consistent with this basic kinetic mechanism.…”

“…All three enzymes in the protein prenyltransferase family require a divalent zinc ion for catalysis, and the amino acids that coordinate zinc are strictly conserved. This conservation, along with a variety of biochemical and structural data, suggests that these enzymes share a common catalytic mechanism where the zinc ion coordinates the sulfur of the peptide substrate (12,26,31,35,50,51). This interaction enhances catalysis by lowering the pK a of the peptide thiol, as well as positioning and, perhaps, activating the thiolate for nucleophilic attack on the isoprenoid diphosphate (26,31,51).…”

“…Initial studies indicated that, similar to FTase, GGTase I required Mg(II) for maximal activity (33,34). However, later studies demonstrated that metal-chelating reagents have little effect on the steadystate turnover rate of GGTase I, suggesting that Mg(II) is not required for maximal activity (35). Sequence alignments and the crystal structures of FTase and GGTase I indicate that the aspartate in FTase that coordinates Mg(II) is replaced by lysine 311 in the ␤-subunit of GGTase I, leading to the suggestion by Beese and colleagues (12,36) that Lys-␤311 in GGTase I replaces the catalytic function of Mg(II) in FTase.…”

Mutagenesis Studies of Protein Farnesyltransferase Implicate Aspartate β352 as a Magnesium Ligand

“…Prenyltransferases contain an active site zinc ion that is required for activity; FTase and GGTase II also use a magnesium ion to facilitate catalysis (16 -19). Surprisingly, GGTase I apparently catalyzes the same prenyl transfer reaction with no dependence on magnesium ions (20).In FTase, the magnesium ion has been proposed to coordinate to the diphosphate of FPP, but the exact location of the magnesium binding site has not yet been identified (21). The bound zinc ion of FTase coordinates to the cysteine sulfur of the protein substrate, lowering the pK a to form a zincthiolate at neutral pH (22, 23).…”

“…Prenyltransferases contain an active site zinc ion that is required for activity; FTase and GGTase II also use a magnesium ion to facilitate catalysis (16 -19). Surprisingly, GGTase I apparently catalyzes the same prenyl transfer reaction with no dependence on magnesium ions (20).…”