Mutagenesis studies of protein farnesyltransferase implicate aspartate beta 352 as a magnesium ligand.
Protein farnesyltransferase (FTase) catalyzes the addition of a farnesyl chain onto the sulfur of a C-terminal cysteine of a protein substrate. Magnesium ions enhance farnesylation catalyzed by FTase by several hundred-fold, with a KMg value of 4 mM. The magnesium ion is proposed to coordinate the diphosphate leaving group of farnesyldiphosphate (FPP) to stabilize the developing charge in the farnesylation transition state. Here we further investigate the magnesium binding site using mutagenesis and biochemical studies. Free FPP binds Mg2+ with a Kd of 120 microM. The 10-fold weaker affinity for Mg2+ observed for the FTase.FPP.peptide ternary complex is probably caused by the positive charges in the diphosphate binding pocket of FTase. Furthermore, mutation of aspartate beta 352 to alanine (D beta 352A) or lysine (D beta 352K) in FTase drastically alters the Mg2+ dependence of FTase catalysis without dramatically affecting the rate constant of farnesylation minus magnesium or the binding affinity of either substrate. In D beta 352A FTase, the KMg increases 28-fold to 110 +/- 30 mM, and the farnesylation rate constant at saturating Mg2+ decreases 27-fold to 0.30 +/- 0.05 s-1. Substitution of a lysine for Asp-beta 352 removes the magnesium activation of farnesylation catalyzed by FTase but does not significantly enhance the rate constant for farnesylation in the absence of Mg2+. In wild type FTase, Mg2+ can be replaced by Mn2+ with a 2-fold lower KMn (2 mM). These results suggest both that Mg2+ coordinates the side chain carboxylate of Asp-beta 352 and that the role of magnesium in the reaction includes positioning the FPP prior to catalysis.
Pickett
Jennifer S
JS
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Bowers
Katherine E
KE
Fierke
Carol A
CA
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000737:chemistry* / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011106:Polyisoprenyl Phosphates / Q000737:chemistry; D012717:Sesquiterpenes
D008024:Ligands; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D001224:Aspartic Acid; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D008274:Magnesium