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J. Biol. Chem., Vol. 278, Issue 51, 51243-51250, December 19, 2003

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Mutagenesis Studies of Protein Farnesyltransferase Implicate Aspartate 352 as a Magnesium Ligand^*

Jennifer S. Pickett, Katherine E. Bowers¶, and Carol A. Fierke||

From the Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055

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 K_Mg 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 K_d of 120 µM. 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 352 to alanine (D352A) or lysine (D352K) 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 D352A FTase, the K_Mg 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-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 K_Mn (2 mM). These results suggest both that Mg²⁺ coordinates the side chain carboxylate of Asp-352 and that the role of magnesium in the reaction includes positioning the FPP prior to catalysis.

Received for publication, August 20, 2003 , and in revised form, October 7, 2003.

^* This work was supported in part by National Institutes of Health Grant GM40602 (to C. A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by National Institutes of Health Training Grant GM08597.

^¶ Supported by National Institutes of Health Postdoctoral Training Grant F32 CA84757.

^|| To whom correspondence should be addressed: 930 N. University Ave., Ann Arbor, MI 48109-1055. Tel.: 734-936-2678; Fax: 734-647-4865; E-mail: fierke{at}umich.edu.

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