title: Upstream polybasic region in peptides enhances dual specificity for prenylation by both farnesyltransferase and geranylgeranyltransferase type I.
abstract: Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase I) catalyze the attachment of a farnesyl or geranylgeranyl lipid, respectively, near the C-terminus of their protein substrates. FTase and GGTase I differ in both their substrate specificity and magnesium dependence, where the activity of FTase, but not GGTase I, is activated by magnesium. Many protein substrates of these enzymes contain an upstream polybasic region that is proposed to increase the affinity of the substrate and aid in plasma membrane association. Here, we demonstrate that the addition of an upstream polybasic region to a peptide substrate enhances the binding affinity of FTase approximately 4-fold for the peptide but diminishes the catalytic efficiency of the reaction, reflected by decreases in both the prenylation rate constant and kcat/KM. Specifically, the prenylation rate constant decreases 7-fold at 5 mM MgCl2 for the peptide KKKSKTKCVIM (C-terminal sequence of K-Ras4B) in comparison to TKCVIM. This decrease is accompanied by an alteration in the dependence on magnesium, as the K(Mg) increases from 2.2 +/- 0.1 mM for TKCVIM to 11.5 +/- 0.1 mM for KKKSKTKCVIM. The presence of an upstream polybasic region does not significantly affect GGTase I-catalyzed reactions, as only minimal changes are seen in Kd, kcat/KM, and k(chem) values. Thus, the presence of an upstream polybasic region enhances the dual prenylation of these substrates, by decreasing the catalytic efficiency of farnesylation catalyzed by FTase to a level comparable to that of geranylgeranylation catalyzed by GGTase I.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Hicks
authors.forename: Heather L
authors.initials: HL
authors.lastname: Hartman
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D011485:Protein Binding; D017368:Protein Prenylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D013379:Substrate Specificity; D018631:ras Proteins / Q000378:metabolism
chemical_list: C117307:KRAS protein, human; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; C064911:p21(ras) farnesyl-protein transferase; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D008239:Lysine