Lysine β311 of protein geranylgeranyltransferase type I partially replaces magnesium

Abstract

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 β352 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 13311 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 (kchem = 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 pKα of 6.4 ± 0.1, likely reflecting deprotonation of the peptide thiol. Mutation of lysine β311 in GGTase I to alanine (Kβ311A) or aspartate (Kβ311D) decreases the kchem 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 Kβ311A and Kβ311D GGTase I 2-5-fold compared with wild type GGTase I with KMg of 140 ± 10 mM and 6.4 ± 0.8 mM, respectively. These results demonstrate that lysine β311 of GGTase I partially replaces the catalytic function of Mg(II) observed in FTase.