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Lysine β311 of Protein Geranylgeranyltransferase Type I Partially Replaces Magnesium

2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês

10.1074/jbc.m403469200

ISSN

1083-351X

Autores

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

Tópico(s)

RNA and protein synthesis mechanisms

Resumo

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 β311 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 pKa 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. 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 β311 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 pKa 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. Prenylation is a type of post-translational modification where a lipid group from either farnesyl diphosphate (FPP) 1The abbreviations used are: FPP, farnesyl diphosphate; Bes, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine; CaaX, tetrapeptide sequence cysteine-aliphatic amino acid-aliphatic amino acid-X (serine glutamine, or methionine for FTase; leucine or phenylalanine for GGTase I); dansylated GCVLL, dansylated pentapeptide Gly-Cys-Val-Leu-Leu; FTase, protein farnesyltransferase; GGPP, geranylgeranyl diphosphate; GGTase I and II, protein geranylgeranyltransferase type I and II; Heppso, N-[2-hydroxyethyl]-piperazine-N′-[hydroxypropanesulfonic acid]; TCEP, tris(2-carboxyethyl)phosphine hydrochloride.1The abbreviations used are: FPP, farnesyl diphosphate; Bes, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine; CaaX, tetrapeptide sequence cysteine-aliphatic amino acid-aliphatic amino acid-X (serine glutamine, or methionine for FTase; leucine or phenylalanine for GGTase I); dansylated GCVLL, dansylated pentapeptide Gly-Cys-Val-Leu-Leu; FTase, protein farnesyltransferase; GGPP, geranylgeranyl diphosphate; GGTase I and II, protein geranylgeranyltransferase type I and II; Heppso, N-[2-hydroxyethyl]-piperazine-N′-[hydroxypropanesulfonic acid]; TCEP, tris(2-carboxyethyl)phosphine hydrochloride. or geranylgeranyl diphosphate (GGPP) is covalently attached via a thioether linkage to a conserved cysteine residue near the carboxyl terminus of a protein (1Farnsworth C.C. 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The attached lipid mediates membrane association and specific protein-protein interactions, which are obligatory for proper functioning of the modified protein (9Casey P.J. Curr. Opin. Cell Biol. 1994; 6: 219-225Crossref PubMed Scopus (169) Google Scholar, 10Marshall C.J. Science. 1993; 259: 1865-1866Crossref PubMed Scopus (302) Google Scholar). Protein farnesyltransferase (FTase), protein geranylgeranyltransferase type I (GGTase I), and protein geranylgeranyltransferase type II (GGTase II) comprise the class of zinc-catalyzed sulfur alkylation enzymes known as the protein prenyltransferases. All three protein prenyltransferases are α/β heterodimers; FTase and GGTase I share common α-subunits and possess β-subunits with 25% sequence identity (8Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1729) Google Scholar, 11Casey P.J. Seabra M.C. J. Biol. Chem. 1996; 271: 5289-5292Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar, 12Taylor J.S. Reid T.S. Terry K.L. Beese L.S. EMBO J. 2003; 22: 5963-5974Crossref PubMed Scopus (109) Google Scholar). FTase and GGTase I prenylate proteins or peptides containing carboxyl-terminal CaaX (C refers to a conserved cysteine residue, a refers to an aliphatic amino acid, and X generally refers to leucine or phenylalanine for GGTase I, and methionine, serine, alanine, or glutamine for FTase) sequences (13Casey P.J. Thissen J.A. Moomaw J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8631-8635Crossref PubMed Scopus (156) Google Scholar, 14Yokoyama K. Goodwin G.W. Ghomashchi F. Gelb M.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5302-5306Crossref PubMed Scopus (217) Google Scholar, 15Moores S.L. Schaber M.D. Mosser S.D. Rands E. O'Hara M.B. Garsky V.M. Marshall M.S. Gibbs J.B. J. Biol. Chem. 1991; 266: 14603-14610Abstract Full Text PDF PubMed Google Scholar, 16Reiss Y. Seabra M.C. 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Sternweis P.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5873-5877Crossref PubMed Scopus (200) Google Scholar, 22Kawata M. Farnsworth C.C. Yoshida Y. Gelb M.H. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8960-8964Crossref PubMed Scopus (111) Google Scholar, 23Yamane H.K. Farnsworth C.C. Xie H.Y. Evans T. Howald W.N. Gelb M.H. Glomset J.A. Fung B.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 286-290Crossref PubMed Scopus (94) Google Scholar). In a fashion distinct from FTase and GGTase I, GGTase II catalyzes the attachment of two geranylgeranyl groups to members of the Rab family of GTPases that contain carboxyl-terminal sequences of XXCC, XCXC, XCCX, and CCXX (C refers to a conserved cysteine residue, and X refers to any amino acid) (24Liang P.H. Ko T.P. Wang A.H. Eur. J. Biochem. 2002; 269: 3339-3354Crossref PubMed Scopus (357) Google Scholar). Recently, much of the work on prenyltransferases has focused on FTase and the protein substrate, Ras, a small GTPase in the receptor tyrosine kinase signaling pathway that is a key regulator of cell division (25Sebti S.M. Hamilton A.D. Farnesyltransferase Inhibitors in Cancer Therapy. Humana Press Inc., Totowa, NJ2001: 21-36Google Scholar). 30% of all human cancers can be linked to mutations in the ras gene, which lead to constitutively activated Ras signaling (25Sebti S.M. Hamilton A.D. Farnesyltransferase Inhibitors in Cancer Therapy. Humana Press Inc., Totowa, NJ2001: 21-36Google Scholar). The post-translational attachment of a farnesyl group by FTase is required for the transforming activity of Ras oncoproteins (25Sebti S.M. Hamilton A.D. Farnesyltransferase Inhibitors in Cancer Therapy. Humana Press Inc., Totowa, NJ2001: 21-36Google Scholar). These observations prompted a search for FTase inhibitors as novel anticancer agents (25Sebti S.M. Hamilton A.D. Farnesyltransferase Inhibitors in Cancer Therapy. Humana Press Inc., Totowa, NJ2001: 21-36Google Scholar). Subsequent research demonstrated that the form of Ras most often mutated in human cancers is K-Ras4B, a protein substrate that can be farnesylated by FTase as well as geranylgeranylated by GGTase I (25Sebti S.M. Hamilton A.D. Farnesyltransferase Inhibitors in Cancer Therapy. Humana Press Inc., Totowa, NJ2001: 21-36Google Scholar). Moreover, geranylgeranylated G proteins in addition to Ras have been shown to play important roles in smooth muscle proliferation and apoptosis (25Sebti S.M. Hamilton A.D. Farnesyltransferase Inhibitors in Cancer Therapy. Humana Press Inc., Totowa, NJ2001: 21-36Google Scholar). The latter observations have initiated a search for GGTase I inhibitors for use in cancer therapy as well as for the treatment of cardiovascular disease (25Sebti S.M. Hamilton A.D. Farnesyltransferase Inhibitors in Cancer Therapy. Humana Press Inc., Totowa, NJ2001: 21-36Google Scholar). The protein prenyltransferases catalyze thioether bond formation between the cysteine sulfur of the protein substrate and the carbon-1 of the isoprenoid substrate, a reaction requiring a catalytically essential zinc ion (13Casey P.J. Thissen J.A. Moomaw J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8631-8635Crossref PubMed Scopus (156) Google Scholar, 14Yokoyama K. Goodwin G.W. Ghomashchi F. Gelb M.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5302-5306Crossref PubMed Scopus (217) Google Scholar, 15Moores S.L. Schaber M.D. Mosser S.D. Rands E. O'Hara M.B. Garsky V.M. Marshall M.S. Gibbs J.B. J. Biol. Chem. 1991; 266: 14603-14610Abstract Full Text PDF PubMed Google Scholar, 16Reiss Y. Seabra M.C. Armstrong S.A. Slaughter C.A. Brown M.S. J. Biol. Chem. 1991; 266: 10672-10677Abstract Full Text PDF PubMed Google Scholar, 17Omer C.A. Kral A.M. Diehl R.E. Prendergast G.C. Powers S. Allen C.M. Kohl N.E. Biochemistry. 1993; 32: 5167-5176Crossref PubMed Scopus (155) Google Scholar, 18Caplin B.E. Hettich L.A. Marshall M.S. Biochim. Biophys. Acta. 1994; 1205: 39-48Crossref PubMed Scopus (62) Google Scholar). Zhang and Casey (8Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1729) Google Scholar) demonstrated that Zn(II) in GGTase I is required for peptide but not isoprenoid binding, suggesting that Zn(II) plays a similar role in both FTase and GGTase I. The zinc ion in FTase coordinates the sulfur of the cysteine within the CaaX sequence of the protein substrate, stabilizing the nucleophilic thiolate (26Hightower K.E. Huang C.C. Fierke C.A. Biochemistry. 1998; 37: 15555-15562Crossref PubMed Scopus (98) Google Scholar). FTase and GGTase II also require Mg(II) for maximal activity (27Moomaw J.F. Casey P.J. J. Biol. Chem. 1992; 267: 17438-17443Abstract Full Text PDF PubMed Google Scholar, 28Reiss Y. Brown M.S. Goldstein J.L. J. Biol. Chem. 1992; 267: 6403-6408Abstract Full Text PDF PubMed Google Scholar). In FTase, Mg(II) accelerates the chemical step 700-fold by coordinating the diphosphate group of the isoprenoid substrate, thereby stabilizing the negative charge buildup on the diphosphate leaving group (29Bowers K.E. Fierke C.A. Biochemistry. 2004; 43: 5256-5265Crossref PubMed Scopus (24) Google Scholar, 30Huang C.C. Hightower K.E. Fierke C.A. Biochemistry. 2000; 39: 2593-2602Crossref PubMed Scopus (74) Google Scholar, 31Saderholm M.J. Hightower K.E. Fierke C.A. Biochemistry. 2000; 39: 12398-12405Crossref PubMed Scopus (54) Google Scholar). Mutagenesis studies provide evidence that Mg(II) is also coordinated by an aspartate residue in FTase (32Pickett J.S. Bowers K.E. Fierke C.A. J. Biol. Chem. 2003; 278: 51243-51250Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Interestingly, the Mg(II) dependence of GGTase I is not well understood. Initial studies indicated that, similar to FTase, GGTase I required Mg(II) for maximal activity (33Zhang F.L. Moomaw J.F. Casey P.J. J. Biol. Chem. 1994; 269: 23465-23470Abstract Full Text PDF PubMed Google Scholar, 34Yokoyama K. McGeady P. Gelb M.H. Biochemistry. 1995; 34: 1344-1354Crossref PubMed Scopus (113) Google Scholar). However, later studies demonstrated that metal-chelating reagents have little effect on the steadystate turnover rate of GGTase I, suggesting that Mg(II) is not required for maximal activity (35Zhang F.L. Casey P.J. Biochem. J. 1996; 320: 925-932Crossref PubMed Scopus (51) Google Scholar). Sequence alignments and the crystal structures of FTase and GGTase I indicate that the aspartate in FTase that coordinates Mg(II) is replaced by lysine 311 in the β-subunit of GGTase I, leading to the suggestion by Beese and colleagues (12Taylor J.S. Reid T.S. Terry K.L. Beese L.S. EMBO J. 2003; 22: 5963-5974Crossref PubMed Scopus (109) Google Scholar, 36Long S.B. Casey P.J. Beese L.S. Nature. 2002; 419: 645-650Crossref PubMed Scopus (169) Google Scholar) that Lys-β311 in GGTase I replaces the catalytic function of Mg(II) in FTase. Here we characterize the kinetics of wild type GGTase I and examine the role of residue Lys-β311 in catalysis using mutagenesis and biochemical experiments. These data demonstrate that the rate constant for geranylgeranylation catalyzed by GGTase I is slower than the farnesylation rate constant catalyzed by FTase and not enhanced by Mg(II), suggesting differences in the catalytic mechanisms between FTase and GGTase I. When Lys-β311 in GGTase I is replaced by alanine or aspartate, geranylgeranylation is now enhanced by Mg(II), indicating that Lys-β311 is important for blocking activation by Mg(II) in wild type GGTase I. However, when the lysine side chain is removed in Kβ311A GGTase I, the rate constant for geranylgeranylation in the absence of Mg(II) decreases 40-fold, indicating that Lys-β311 modestly enhances catalysis. Furthermore, the lysine side chain is not as effective at stabilizing the diphosphate leaving group as Mg(II) in FTase, which enhances catalysis 700-fold; therefore, GGTase I is not as efficient as FTase in catalyzing prenylation (30Huang C.C. Hightower K.E. Fierke C.A. Biochemistry. 2000; 39: 2593-2602Crossref PubMed Scopus (74) Google Scholar). Miscellaneous Methods—All assays were performed at 25 °C. All curve fitting was performed with Kaleidagraph (Synergy Software, Reading, PA). Tritium-labeled farnesyl diphosphate ([1-3H]FPP) and tritium-labeled geranylgeranyl diphosphate ([1-3H]GGPP) were purchased from Amersham Biosciences. The peptides GCVLL and dansylated GCVLL were synthesized and purified by high pressure liquid chromatography by Bethyl Laboratories (Montgomery, TX). The concentration of peptide was determined spectroscopically at 412 nm by reaction of the cysteine thiol with 5,5′-dithiobis(2-nitrobenzoic acid), using an extinction coefficient of 14,150 m-1 cm-1 (37Riddles P.W. Blakeley R.L. Zerner B. Anal. Biochem. 1979; 94: 75-81Crossref PubMed Scopus (919) Google Scholar). GGTase I concentration was determined by absorbance at 280 nm using an extinction coefficient of 134,000 m-1 cm-1, which was calculated using the method of Edelhoch (38Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (2999) Google Scholar; data not shown). Thin layer chromatography (TLC) plates were pre-run in 100% acetone before use. Subcloning of GGTase I—A bacterial expression vector for rat GGTase I was prepared in the following way. Mutagenesis of the high expression rat FTase pET23a-FPT plasmid (39Zimmerman K.K. Scholten J.D. Huang C.C. Hupe D.J. Protein Expr. Purif. 1998; 14: 395-402Crossref PubMed Scopus (35) Google Scholar) was performed using the QuikChange site-directed mutagenesis kit (Stratagene) to introduce an EcoRI restriction site into the stop/start region between the genes encoding the two subunits of FTase with the following codon changes: GAGGAGTTT to GAGGAATTC. The modified pET23a-FPT plasmid was cut using the restriction enzymes EcoRI and NdeI to remove a DNA fragment containing the β-subunit of FTase. The remaining plasmid fragment was purified from the DNA fragment encoding the FPT β-subunit using a 1.2% agarose gel. The gene encoding the β-subunit of rat GGTase I was amplified from the plasmid pET28a-GGT-1 (33Zhang F.L. Moomaw J.F. Casey P.J. J. Biol. Chem. 1994; 269: 23465-23470Abstract Full Text PDF PubMed Google Scholar, 39Zimmerman K.K. Scholten J.D. Huang C.C. Hupe D.J. Protein Expr. Purif. 1998; 14: 395-402Crossref PubMed Scopus (35) Google Scholar, 40Zhang F.L. Diehl R.E. Kohl N.E. Gibbs J.B. Giros B. Omer C.A. J. Biol. Chem. 1994; 269: 3175-3180Abstract Full Text PDF PubMed Google Scholar; a gift from Patrick Casey, Duke University) using PCR with primers that incorporated an NdeI restriction site at the 5′-end of the β-subunit and an EcoRI restriction site at the 3′-end of the β-subunit. This PCR fragment was purified using a 1.2% agarose gel, digested with the restriction enzymes EcoRI and NdeI, and repurified to obtain the DNA encoding the β-subunit of GGTase I. This DNA fragment was ligated into the NdeI/EcoRI-digested pET23a-FPT plasmid using a ligation premix (Clonables, EMD Biosciences, San Diego) to generate the plasmid pET23a-GGPT. The plasmid was transformed into XL-1 Blue cells, and the cells were grown in Luria-Bertani medium supplemented with 100 mg liter-1 ampicillin. The plasmid was purified using a Plasmid Midi Kit (Qiagen, Valencia, CA). The sequence of the entire gene was confirmed by DNA sequencing (University of Michigan DNA Sequencing Core, Ann Arbor). Preparation of GGTase I—Recombinant rat GGTase I was overexpressed in Escherichia coli BL21(DE3) pET23a-GGPT cells and purified as described below. The cells were grown in rich induction medium (20 g liter-1 tryptone, 10 g liter-1 yeast extract, 5 g liter-1 NaCl, 50 mg liter-1 ampicillin, 1% glucose, 0.06 mm ZnSO4, 0.22 g liter-1 Na2HPO4, 0.11 g liter-1 KH2PO4, 0.036 g liter-1 NH4Cl, pH 7.35) at 37 °C to an A600 of 0.8, induced by the addition of 0.4 mm isopropyl β-d-1-thiogalactopyranoside and 0.5 mm ZnSO4, and incubated at 25 °C for 14 h (39Zimmerman K.K. Scholten J.D. Huang C.C. Hupe D.J. Protein Expr. Purif. 1998; 14: 395-402Crossref PubMed Scopus (35) Google Scholar). The harvested cells were lysed using a microfluidizer (Microfluidics, Newton, MA). The cell lysate from 2 liters of cells was loaded onto a DEAE-Sephacel (Amersham Biosciences, 30-ml bed volume) or DEAE-cellulose (Whatman, 100-ml bed volume) column preequilibrated with HTZ buffer (50 mm HEPES, pH 7.8, 1 mm TCEP, 10 μm ZnCl2). The column was run by gravity with a linear gradient (400 ml) from 0 to 0.5 m NaCl in HTZ buffer. GGTase I eluted at 0.3 m NaCl. The fractions were tested for GGTase I activity using a 96-well plate format. Each well contained 50 mm Heppso, pH 7.8, 5 mm MgCl2, 1 mm TCEP, 0.2 μm GGPP (Sigma), 1 μm dansylated GCVLL, and 10 μl of protein sample (100-μl total volume). The relative fluorescence of each fraction was measured after the addition of dansylated GCVLL by the Polarstar Galaxy fluorescence plate reader (BMG Laboratory Technologies, Durham, NC, λex = 280 nm, λem = 490 nm). Fractions containing GGTase I were pooled and concentrated using Amicon Ultra centrifugal filter devices (Millipore, Bedford, MA). The protein was dialyzed against HTZ buffer overnight, then run over a Q-Sepharose HP column (Amersham Biosciences, 40-ml bed volume) using fast protein liquid chromatography (AKTA Prime, Amersham Biosciences) with a 0–0.5 m NaCl step gradient (400 ml). The active fractions were again pooled and concentrated using Amicon Ultra centrifugal filter devices (Millipore). The protein was then immediately loaded onto a Poros 20 HQ column (Applied Biosystems, Foster City, CA 1.7-ml bed volume) and fractionated using a step gradient at 0.1, 0.5, and 2 m NaCl (90 ml). The prepared GGTase I was determined by SDS-PAGE to be >90% pure. The protein was dialyzed against HT buffer (50 mm HEPES, pH 7.8, 1 mm TCEP), concentrated to 31 μm using Amicon Ultra centrifugal filter devices (Millipore), and frozen at -80 °C. Using these procedures, 15 mg of purified GGTase I was obtained from 1 liter of cell culture. Preparation of Mutant GGTase I—Mutagenesis of the pET23a-GGPT plasmid was performed using the QuikChange site-directed mutagenesis kit with the following codon changes: Kβ311A, AAA to GCA, and Kβ311D, AAA to GAC. The changes were confirmed by DNA sequencing (University of Michigan DNA Sequencing Core, Ann Arbor). Recombinant rat GGTase I mutants were overexpressed in E. coli and purified as described above except that the Q-Sepharose HP column was omitted. Instead, after the DEAE-cellulose column, the active fractions were loaded onto a Poros 20 HQ column (Applied Biosystems, 20.2-ml bed volume) and fractionated with a linear gradient (800 ml) from 0.05 to 0.5 m NaCl in HTZ buffer. The active fractions were loaded immediately onto a second Poros 20 HQ column (Applied Biosystems, 7.9-ml bed volume) and fractionated with a step gradient at 0.1 and 0.5 m NaCl. GGPP Binding Affinity—The affinity of wild type GGTase I for GGPP and FPP and Lys-β311 GGTase I mutants for GGPP was determined by equilibrium dialysis (30Huang C.C. Hightower K.E. Fierke C.A. Biochemistry. 2000; 39: 2593-2602Crossref PubMed Scopus (74) Google Scholar, 39Zimmerman K.K. Scholten J.D. Huang C.C. Hupe D.J. Protein Expr. Purif. 1998; 14: 395-402Crossref PubMed Scopus (35) Google Scholar). A 1-ml solution containing 0.1–100 nm enzyme was preincubated with a constant concentration of [1-3H]GGPP or [1-3H]FPP (5, 10, or 20 nm) for 1 h at room temperature in buffer (50 mm Heppso, pH 7.8, and 1 mm TCEP). This solution was then dialyzed against a 1-ml solution of the same concentration of [1-3H]GGPP or [1-3H]FPP (5, 10, or 20 nm) in the same buffer using a 25,000 molecular weight cutoff dialysis membrane (Spectra/Por 7, Spectrum Laboratories, Rancho Dominguez, CA). After 20 h, the radioactivity in 100-μl samples from both sides of the membrane was quantified in triplicate by scintillation counting and averaged. The fraction of GGPP bound to GGTase I (E:GGPP) was determined by subtracting the counts on the [1-3H]GGPP side from the counts on the enzyme side, then dividing the difference by the total counts. The concentration of E:GGPP was calculated by multiplying the fraction of GGPP bound to E:GGPP by the initial [1-3H]GGPP concentration, [GGPP]total (5–20 nm). The [E]free was calculated by subtracting [E:GGPP] from [E]total. The dissociation constant, KD, was then determined by fitting Equation 1 to the dependence of E:GGPP/GGPPtotal on [E]free where EP refers to the fraction of GGPP bound to E:GGPP at saturation. The data were truncated at [E]free = 5KD.E:GGPPGGPPtotal=EP1+KD/[E]free(Eq. 1) Peptide Binding Affinity—The affinity of GGTase I and Lys-β311 GGTase I mutants for dansylated GCVLL was determined by fluorescence anisotropy (26Hightower K.E. Huang C.C. Fierke C.A. Biochemistry. 1998; 37: 15555-15562Crossref PubMed Scopus (98) Google Scholar, 41Lakowicz J.R. Principles of Fluorescence Spectroscopy. 2nd Ed. Kluwer Academic Publishers, Norwell, MA1999: 291-319Crossref Google Scholar), where the dansyl group of the peptide is excited at 340 nm (band pass = 16 nm), and its emission is monitored at 525 nm (band pass = 16 nm). The samples were prepared with 50 mm Heppso, pH 7.8, 2 mm TCEP, 2 nm dansylated GCVLL, and 10 nm EDTA. The samples were titrated with GGTase I (0–150 nm), and additional dansylated GCVLL was added to maintain the 2 nm concentration of peptide. For comparison, this titration was repeated in the presence of 1 μm 3-azageranylgeranyl diphosphate (42Sagami H. Korenaga T. Ogura K. Steiger A. Coates R.M. Arch. Biochem. Biophys. 1992; 297: 314-320Crossref PubMed Scopus (24) Google Scholar, a gift from Robert Coates, University of Illinois), a nonhydrolyzable GGPP analog. As a control, both titrations were repeated using 2 nm iodoacetamide-treated dansylated GCVLL (which should not bind to GGTase I). All samples were incubated for 5 min without stirring prior to each measurement at 25 °C. A weighted fit of Equation 2 to the data yields the apparent dissociation constants where ΔA represents the observed fluorescence anisotropy at 525 nm corrected for background, EP is the fluorescence anisotropy end point, IF is the initial fluorescence anisotropy, [enzyme] is the concentration of GGTase I, and KDpeptide is the dissociation constant for dansylated GCVLL.ΔA=EP1+KDpeptide/[enzyme]+IF(Eq. 2) Transient Kinetics—Single turnover assays were performed for wild type and mutant GGTase I at multiple magnesium concentrations (0.0006–175 mm) in 50 mm Heppso, pH 7.8, 2 mm TCEP, with ionic strength kept constant at 0.2 m with NaCl up to 60 mm Mg(II) as described previously (31Saderholm M.J. Hightower K.E. Fierke C.A. Biochemistry. 2000; 39: 12398-12405Crossref PubMed Scopus (54) Google Scholar). Reactions with prenylation rate constants less than 0.1 s-1 were performed manually using 0.8 μm GGTase I, 0.4 μm [1-3H]FPP or [1-3H]GGPP, and 100 μm peptide GCVLL (8-μl reaction volume). GGTase I was preincubated with [1-3H]FPP or [1-3H]-GGPP for 15 min at room temperature; the reaction was subsequently initiated by the addition of peptide. The reactions were quenched at varying times (3 s–1 h) by the addition of 8 μl of cold 80% isopropyl alcohol, 20% acetic acid (v/v) and placed on ice. For reactions with rate constants faster than 0.1 s-1, a KinTek rapid quench apparatus was used (KinTek Corporation, Austin, TX). The 30-μl reactions contained 0.8 μm GGTase I, 0.4 μm [1-3H]FPP or [1-3H]GGPP, and 100 μm peptide. The reactions were quenched with 80% isopropyl alcohol, 20% acetic acid at varying times (0.05–120 s), then dried under vacuum and resuspended in 50% isopropyl alcohol. The product was separated from substrate by TLC on polyester-backed silica gel plates (Whatman PE SIL G) with an 8:1:1 (v/v/v) isopropyl alcohol/NH4OH/H2O mobile phase. The product migrates in this mobile phase, but the GGPP substrate remains at the origin, so the plates were cut accordingly, and the radioactivity was quantified by scintillation counting. The radioactivity in the product was divided by the total radioactivity for each time point to calculate the fraction of product formed. The rate constant for product formation (kobs) was determined by fitting Equation 3 to the data, where Pt is the fraction product formed at time t, and P∞ is the calculated reaction end point, which varied from 60 to 90%.PtP∞=1−e−kobs*t(Eq. 3) The magnesium dependence was determined by measuring the single turnover rate constant, kobs, as a function of Mg(II) concentration. A weighted fit of Equation 4 to these data was used to determine: KMg, the apparent dissociation constant; kmaxMg, the rate constant of the reaction at saturating Mg(II) concentration; and k0, the rate constant of the reaction in the absence of magnesium.kobs=kmaxMg1+KMg/[Mg(II)]+k0(Eq. 4) The pH dependence of the single turnover rate constant was measured in 50 mm Bes (pH 6.1–7.0), 50 mm Heppso, pH 7.8–8.0, or 50 mm Bicine, pH 8.3–9.0; 2 mm TCEP was included as a reductant. Reactions were performed manually using 0.8 μm GGTase I, 0.4 μm [1-3H]GGPP, and 100 μm peptide (8-μl reaction volume) and analyzed as described above. The pKa of the react

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