Influence of metal ions on substrate binding and catalytic activity of mammalian protein geranylgeranyltransferase type-I.
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.
Zhang
F L
FL
Department of Molecular Cancer Biology, Duke University Medical Center, Durham, NC 27710-3686, USA.
Casey
P J
PJ
D019883:Alkyl and Aryl Transferases; D002104:Cadmium / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D003619:Dansyl Compounds / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051231:Farnesyltranstransferase; D005453:Fluorescence; D008274:Magnesium / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D010455:Peptides / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects*; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D002614:Chelating Agents; D003619:Dansyl Compounds; D004791:Enzyme Inhibitors; D008670:Metals; D010455:Peptides; D010618:Phenanthrolines; D011106:Polyisoprenyl Phosphates; D002104:Cadmium; D004492:Edetic Acid; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D051231:Farnesyltranstransferase; D008274:Magnesium; D015032:Zinc; C002963:geranylgeranyl pyrophosphate; C025205:1,10-phenanthroline