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J. Biol. Chem., Vol. 279, Issue 29, 30546-30553, July 16, 2004

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Lysine 311 of Protein Geranylgeranyltransferase Type I Partially Replaces Magnesium^*

Heather L. Hartman, Katherine E. Bowers, and Carol A. Fierke

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

Protein geranylgeranyltransferase type I (GGTase I) catalyzes the attachment of a geranylgeranyl lipid group near the carboxyl terminus of protein substrates. Unlike protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type II, which require both Zn(II) and Mg(II) for maximal turnover, GGTase I turnover is dependent only on Zn(II). In FTase, the magnesium ion is coordinated by aspartate 352 and the diphosphate of farnesyl diphosphate to stabilize the developing charge in the transition state (Pickett, J. S., Bowers, K. E., and Fierke, C. A. (2003) J. Biol. Chem. 278, 51243–51250). In GGTase I, lysine 311 is substituted for this aspartate and is proposed to replace the catalytic function of Mg(II) (Taylor, J. S., Reid, T. S., Terry, K. L., Casey, P. J., and Beese, L. S. (2003) EMBO J. 22, 5963–5974). Here we demonstrate that the prenylation rate constant catalyzed by wild type GGTase I (k_chem = 0.18 ± 0.02 s^-1) is not dependent on Mg(II), is 20-fold slower than the maximal rate constant catalyzed by FTase, and has a single pK_a of 6.4 ± 0.1, likely reflecting deprotonation of the peptide thiol. Mutation of lysine 311 in GGTase I to alanine (K311A) or aspartate (K311D) decreases the k_chem in the absence of magnesium 9–41-fold without significantly affecting the binding affinity of either substrate. Furthermore, the geranylgeranylation rate constant is enhanced by the addition of Mg(II) for K311A and K311D GGTase I 2–5-fold compared with wild type GGTase I with K_Mg of 140 ± 10 mM and 6.4 ± 0.8 mM, respectively. These results demonstrate that lysine 311 of GGTase I partially replaces the catalytic function of Mg(II) observed in FTase.

Received for publication, March 29, 2004 , and in revised form, May 5, 2004.

^* This work was supported by National Institutes of Health Grant GM40602 (to C. A. F.). Partial funding was also provided by National Institutes of Health Postdoctoral Fellowship F32 CA84757 (to K. E. B.), Gaining Assistance in Areas of National Need Grant 037733 (to H. L. H.), and NIGMS National Institutes of Health Training Grant GM07767 (to H. L. H.). 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.

Present address: Regeneron Pharmaceuticals, Tarrytown, NY 10591-6707.

To whom correspondence should be addressed: Dept. of Chemistry, University of Michigan, 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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