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Mutational Analysis of Conserved Residues of the ॆ-Subunit of Human Farnesyl:Protein Transferase

1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês

10.1074/jbc.272.43.27319

1083-351X

Astrid M. Kral, Ronald E. Diehl, S. J. DESOLMS, Theresa M. Williams, Nancy E. Kohl, Charles A. Omer,

Biotin and Related Studies

The roles of 11 conserved amino acids of the ॆ-subunit of human farnesyl:protein transferase (FTase) were examined by performing kinetic and biochemical analyses of site-directed mutants. This biochemical information along with the x-ray crystal structure of rat FTase indicates that residues His-248, Arg-291, Lys-294, and Trp-303 are involved with binding and utilization of the substrate farnesyl diphosphate. Our data confirm structural evidence that amino acids Cys-299, Asp-297, and His-362 are ligands for the essential Zn2+ ion and suggest that Asp-359 may also play a role in Zn2+ binding. Additionally, we demonstrate that Arg-202 is important for binding the essential C-terminal carboxylate of the protein substrate. The roles of 11 conserved amino acids of the ॆ-subunit of human farnesyl:protein transferase (FTase) were examined by performing kinetic and biochemical analyses of site-directed mutants. This biochemical information along with the x-ray crystal structure of rat FTase indicates that residues His-248, Arg-291, Lys-294, and Trp-303 are involved with binding and utilization of the substrate farnesyl diphosphate. Our data confirm structural evidence that amino acids Cys-299, Asp-297, and His-362 are ligands for the essential Zn2+ ion and suggest that Asp-359 may also play a role in Zn2+ binding. Additionally, we demonstrate that Arg-202 is important for binding the essential C-terminal carboxylate of the protein substrate. Farnesyl:protein transferase (FTase) 1The abbreviations used in the manuscript are FTase, farnesyl:protein transferase; GGTase-I, geranylgeranyl:protein transferase type I; GGTase-II, geranylgeranyl:protein transferase type II; PTase, prenyl:protein transferase; FTI, FTase inhibitor; CAAX, tetrapeptide in which C = Cys, A is usually an aliphatic amino acid, X = any amino acid; FPP, farnesyl diphosphate; Ras-CVLS, yeast Ras1[Leu-68](term.)-SLKCVLS; SPA, scintillation proximity assay; DTT, dithiothreitol. 1The abbreviations used in the manuscript are FTase, farnesyl:protein transferase; GGTase-I, geranylgeranyl:protein transferase type I; GGTase-II, geranylgeranyl:protein transferase type II; PTase, prenyl:protein transferase; FTI, FTase inhibitor; CAAX, tetrapeptide in which C = Cys, A is usually an aliphatic amino acid, X = any amino acid; FPP, farnesyl diphosphate; Ras-CVLS, yeast Ras1[Leu-68](term.)-SLKCVLS; SPA, scintillation proximity assay; DTT, dithiothreitol. catalyzes thioether bond formation between farnesyl, from farnesyl diphosphate (FPP), and the sulfur atom of a cysteine residue near the C terminus of its protein substrate (1Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Cell. 1990; 62: 81-88Abstract Full Text PDF PubMed Scopus (701) Google Scholar, 2Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1727) Google Scholar). The protein substrate cysteine residue is located within a C-terminal motif called a CAAX box in which C is the cysteine that is S-farnesylated, A is often an aliphatic amino acid, and X is methionine, serine, glutamine, cysteine, or alanine (1Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Cell. 1990; 62: 81-88Abstract Full Text PDF PubMed Scopus (701) Google Scholar, 3Moores S.L. Schaber M.D. Mosser S.D. Rands E. O'Hara M.B. Garsky V.M. Marshall M.S. Pompliano D.L. Gibbs J.B. J. Biol. Chem. 1991; 266: 14603-14610Abstract Full Text PDF PubMed Google Scholar, 4Reiss Y. Stradley S.J. Gierasch L.M. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 732-736Crossref PubMed Scopus (310) Google Scholar). S-Farnesylation is the first step by which a number of proteins, including all forms of the Ras proto-oncoprotein, are post-translationally lipid-modified, facilitating membrane association and in some cases protein-protein interaction (2Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1727) Google Scholar). Membrane association is required for Ras function, thus inhibitors of FTase have been proposed as antitumor agents (5Gibbs J.B. Oliff A. Kohl N.E. Cell. 1994; 77: 175-178Abstract Full Text PDF PubMed Scopus (509) Google Scholar). FTase inhibitors (FTIs) have been shown to inhibit anchorage-independent growth of ras-transformed rodent fibroblasts and human tumor cell lines (6Nagasu T. Yoshimatsu K. Rowill C. Lewis M.D. Garcia A.M. Cancer Res. 1995; 55: 5310-5314PubMed Google Scholar, 7Kohl N.E. Mosser S.D. deSolms S.J. Giuliani E.A. Pompliano D.L. Graham S.L. Smith R.L. Scolnick E.M. Oliff A. Gibbs J.B. Science. 1993; 260: 1934-1937Crossref PubMed Scopus (618) Google Scholar, 8Sepp-Lorenzino L. Ma Z. Rands E. Kohl N.E. Gibbs J.B. Oliff A. Rosen N. Cancer Res. 1995; 55: 5302-5309PubMed Google Scholar). In addition, FTIs have shown antitumor effects in rodents (6Nagasu T. Yoshimatsu K. Rowill C. Lewis M.D. Garcia A.M. Cancer Res. 1995; 55: 5310-5314PubMed Google Scholar, 9Kohl N.E. Omer C.A. Conner M.W. Anthony N.J. Davide J.P. deSolms S.J. Giuliani E.A. Gomez R.P. Graham S.L. Hamilton K. Handt L.K. Hartman G.D. Koblan K.S. Kral A.M. Miller P.J. Mosser S.D. O'Neill T.J. Rands E. Schaber M.D. Gibbs J.B. Oliff A. Nat. Genet. 1995; 1: 792-797Crossref Scopus (510) Google Scholar). FTase kinetically proceeds through an ordered, sequential mechanism in which FPP is the first substrate bound (10Pompliano D.L. Schaber M.D. Mosser S.D. Omer C.A. Shafer J.A. Gibbs J.B. Biochemistry. 1993; 32: 8341-8347Crossref PubMed Scopus (109) Google Scholar, 11Furfine E.S. Leban J.J. Landavazo A. Moomaw J.F. Casey P.J. Biochemistry. 1995; 34: 6857-6862Crossref PubMed Scopus (161) Google Scholar). Subsequent protein substrate binding to FTase depends on an enzyme-bound Zn2+ion (12Reiss Y. Brown M.S. Goldstein J.L. J. Biol. Chem. 1992; 267: 6403-6408Abstract Full Text PDF PubMed Google Scholar, 13Chen W.-J. Moomaw J.F. Overton L. Kost T.A. Casey P.J. J. Biol. Chem. 1993; 268: 9675-9680Abstract Full Text PDF PubMed Google Scholar). Recent spectral data indicate that upon formation of the FTase·FPP·CAAX ternary complex, the cysteine thiol of the CAAX sequence interacts directly with Zn2+, possibly forming a thiolate anion (14Huang C.-C. Casey P.J. Fierke C.A. J. Biol. Chem. 1997; 272: 20-23Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In addition to the enzyme-bound Zn2+, FTase activity requires millimolar Mg2+ (12Reiss Y. Brown M.S. Goldstein J.L. J. Biol. Chem. 1992; 267: 6403-6408Abstract Full Text PDF PubMed Google Scholar). Two additional prenyl:protein transferases (PTases) have been described that catalyze reactions similar to that of FTase. Geranylgeranyl:protein transferase type I (GGTase-I) geranylgeranylates protein substrates with a C-terminal CAAX motif in whichX is usually leucine (3Moores S.L. Schaber M.D. Mosser S.D. Rands E. O'Hara M.B. Garsky V.M. Marshall M.S. Pompliano D.L. Gibbs J.B. J. Biol. Chem. 1991; 266: 14603-14610Abstract Full Text PDF PubMed Google Scholar, 15Moomaw J.F. Casey P.J. J. Biol. Chem. 1992; 267: 17438-17443Abstract Full Text PDF PubMed Google Scholar). Geranylgeranyl:protein transferase type II (GGTase-II, also known as Rab geranylgeranyltransferase) geranylgeranylates both cysteine residues in protein substrates with Cys-Cys, Cys-Xaa-Cys, and Cys-Cys-Xaa-Xaa C-terminal motifs (3Moores S.L. Schaber M.D. Mosser S.D. Rands E. O'Hara M.B. Garsky V.M. Marshall M.S. Pompliano D.L. Gibbs J.B. J. Biol. Chem. 1991; 266: 14603-14610Abstract Full Text PDF PubMed Google Scholar, 16Seabra M.C. Goldstein J.L. Sudhof T.C. Brown M.S. J. Biol. Chem. 1992; 267: 14497-14503Abstract Full Text PDF PubMed Google Scholar). PTases have been found in yeast, mammals, and plants and appear to be ubiquitously expressed in eukaryotes (2Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1727) Google Scholar,17Schmitt D. Cullan K. Gruissem W. Plant Physiol. 1996; 112: 767-777Crossref PubMed Scopus (23) Google Scholar). The catalytic moiety of all PTases purified to date are αॆ heterodimers, although GGTase-II requires a third subunit for protein substrate presentation (15Moomaw J.F. Casey P.J. J. Biol. Chem. 1992; 267: 17438-17443Abstract Full Text PDF PubMed Google Scholar, 16Seabra M.C. Goldstein J.L. Sudhof T.C. Brown M.S. J. Biol. Chem. 1992; 267: 14497-14503Abstract Full Text PDF PubMed Google Scholar, 18Reiss Y. Seabra M.C. Armstrong S.A. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1991; 266: 10672-10677Abstract Full Text PDF PubMed Google Scholar). The α-subunits of FTase and GGTase-I are identical and show similarity to the corresponding subunit of GGTase-II (19Seabra M.C. Reiss Y. Casey P.J. Brown M.S. Goldstein J.L. Cell. 1991; 65: 429-434Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 20Zhang F.L. Diehl R.E. Kohl N.E. Gibbs J.B. Giros B. Casey P.J. Omer C.A. J. Biol. Chem. 1994; 269: 3175-3180Abstract Full Text PDF PubMed Google Scholar, 21Armstrong S.A. Seabra M.C. Sudhof T.C. Goldstein J.L. Brown M. J. Biol. Chem. 1993; 268: 12221-12229Abstract Full Text PDF PubMed Google Scholar). The ॆ-subunits for each of these enzymes are distinct, but have overall amino acid similarity of approximately 307 (20Zhang F.L. Diehl R.E. Kohl N.E. Gibbs J.B. Giros B. Casey P.J. Omer C.A. J. Biol. Chem. 1994; 269: 3175-3180Abstract Full Text PDF PubMed Google Scholar, 21Armstrong S.A. Seabra M.C. Sudhof T.C. Goldstein J.L. Brown M. J. Biol. Chem. 1993; 268: 12221-12229Abstract Full Text PDF PubMed Google Scholar). In cross-linking studies, substrate proteins and photoactivatable analogs of FPP and CAAX peptides have been shown to bind to the ॆ-subunit of FTase (18Reiss Y. Seabra M.C. Armstrong S.A. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1991; 266: 10672-10677Abstract Full Text PDF PubMed Google Scholar, 22Omer C.A. Kral A.M. Diehl R.E. Prendergast G.C. Powers S. Allen C.M. Gibbs J.B. Kohl N.E. Biochemistry. 1993; 32: 5167-5176Crossref PubMed Scopus (155) Google Scholar, 23Ying W. Sepp-Lorenzino L. Cai K. Aloise P. Coleman P.S. J. Biol. Chem. 1994; 269: 470-477Abstract Full Text PDF PubMed Google Scholar). Similarly, photoactivatable geranylgeranyl diphosphate analogs can be cross-linked to the ॆ-subunit of GGTase-I (24Bukhtiyarov Y.E. Omer C.A. Allen C.M. J. Biol. Chem. 1995; 270: 19035-19040Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 25Yokoyama K. McGeady P. Gelb M.H. Biochemistry. 1995; 34: 1344-1354Crossref PubMed Scopus (113) Google Scholar). The recently published x-ray crystal structure of rat FTase (>937 amino acid sequence identity to human FTase) identified three conserved amino acids of the ॆ-subunit as ligands for the essential Zn2+ ion (26Park J.T. Boduluri S.R. Moomaw J.F. Casey P.J. Beese L.T. Science. 1997; 275: 1800-1804Crossref PubMed Scopus (329) Google Scholar). Thus the ॆ-subunit appears to play key roles in substrate binding and presumably in catalysis. In this communication we have examined the role of the human FTase ॆ-subunit by analyzing site-directed mutants of amino acids Arg-202, His-248, Cys-254, Arg-291, Lys-294, Asp-297, Cys-299, Tyr-300, Trp-303, Asp-359, and His-362. These residues are conserved among the ॆ-subunits of all PTase enzymes identified to date and are near the essential Zn2+ ion in rat FTase (26Park J.T. Boduluri S.R. Moomaw J.F. Casey P.J. Beese L.T. Science. 1997; 275: 1800-1804Crossref PubMed Scopus (329) Google Scholar). We present biochemical evidence indicating the involvement of these amino acids in substrate interaction, zinc binding, and catalysis. Utilizing these results and the rat FTase crystal structure, we propose that ॆ-subunit residues Arg-291, Lys-294, Trp-303, and possibly His-248 are involved in FPP binding. Our data suggest that ॆ-subunit residue Arg-202 interacts with the C-terminal carboxylate of the CAAX substrate. We confirm that ॆ-subunit residues Asp-297, Cys-299, and His-362 are Zn2+ ligands and present data indicating that Asp-359 may also have an effect on Zn2+ binding. The protein substrate used in this study wasSaccharomyces cerevisiae Ras1 [Leu-68](term)-SLKCVLS and is referred to as Ras-CVLS (3Moores S.L. Schaber M.D. Mosser S.D. Rands E. O'Hara M.B. Garsky V.M. Marshall M.S. Pompliano D.L. Gibbs J.B. J. Biol. Chem. 1991; 266: 14603-14610Abstract Full Text PDF PubMed Google Scholar). It was expressed and purified as described previously (3Moores S.L. Schaber M.D. Mosser S.D. Rands E. O'Hara M.B. Garsky V.M. Marshall M.S. Pompliano D.L. Gibbs J.B. J. Biol. Chem. 1991; 266: 14603-14610Abstract Full Text PDF PubMed Google Scholar). ENTENSIFYTM and [3H]FPP (22.5 Ci/mmol) were purchased from NEN Life Science Products. Unlabeled FPP was obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO). The biotinylation kit, neutral density scintillation proximity assay (SPA) streptavidin beads, and65ZnCl2 (10.3 Ci/mmol) were purchased from Amersham. Bradford protein determination reagents and Bio-Spin-6 columns were acquired from Bio-Rad. 67 polyacrylamide gels (7 × 8 × 0.1 cm) in 89 mm Tris borate, pH 8.3, 0.1 mm EDTA (TBE) were purchased from Novex (San Diego, CA). X-ray film (X-Omat AR) was obtained from Kodak. Wild type human FTase was expressed from pRD517 in Escherichia coli BL21 (DE3). pRD517 is similar to pFPTase-ॆαopt1 (22Omer C.A. Kral A.M. Diehl R.E. Prendergast G.C. Powers S. Allen C.M. Gibbs J.B. Kohl N.E. Biochemistry. 1993; 32: 5167-5176Crossref PubMed Scopus (155) Google Scholar) except that it has a bacteriophage T7 promoter driving expression of the two subunits of FTase. The coding sequences for the ॆ- and α-subunits of human FTase were cloned in tandem into BamHI + HindIII cleaved pT5T downstream of the T7 promoter (27Eisenberg S.P. Evans R.J. Arend W.P. Verderber E. Brewer M.T. Hannum C.H. Thompson R.C. Nature. 1990; 343: 341-346Crossref PubMed Scopus (920) Google Scholar). The sequence of the insert is shown in Sequence 1. Recombinant, human FTase made from this strain has the epitope Glu-Glu-Phe attached to the C terminus of the ॆ-subunit to facilitate purification (22Omer C.A. Kral A.M. Diehl R.E. Prendergast G.C. Powers S. Allen C.M. Gibbs J.B. Kohl N.E. Biochemistry. 1993; 32: 5167-5176Crossref PubMed Scopus (155) Google Scholar). Site-directed mutations were introduced into pRD517 using the polymerase chain reaction (28Higuchi R. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego1990: 177-183Google Scholar). Mutationally silent restriction endonuclease sites were also introduced during the mutagenesis enabling us to screen clones for the presence of the new site. The amino acid changes made are abbreviated using the following notation. 舠ॆR202A舡 indicates that ॆ-subunit residue arginine 202 was changed to alanine. The corresponding heterodimeric human FTase is 舠αॆR202A.舡 All mutated regions were sequenced to ensure that only the intended changes were made. Bacterial growth and induction of FTase synthesis with isopropyl-1-thio-ॆ-d-galactopyranoside was performed as described (29Omer C.A. Diehl R.E. Kral A.M. Methods Enzymol. 1995; 250: 3-12Crossref PubMed Scopus (16) Google Scholar). Purification of the recombinant FTases was similar to that described previously (29Omer C.A. Diehl R.E. Kral A.M. Methods Enzymol. 1995; 250: 3-12Crossref PubMed Scopus (16) Google Scholar). Briefly, E. coli were lysed by sonication, and the debris was pelleted. The soluble protein was applied to a YL1/2 antibody column to bind to the Glu-Glu-Phe C-terminal epitope on the FTase ॆ-subunit. Specifically bound protein was eluted at neutral pH with 5 mm Asp-Phe (Sigma). Approximately a 10-fold molar excess of ZnCl2 was added to the enzyme before final purification by chromatography on Mono Q (Pharmacia Biotech Inc.). FTase was the only major absorbance (A 280 nm) peak seen eluting from the Mono Q column. The final enzyme preparations were >707 pure as determined by SDS-polyacrylamide gel electrophoresis and subsequent Coomassie Blue staining. Since this purification method does not require following the enzyme activity, we could isolate mutant enzymes with little residual catalytic activity. FTase activity was assayed in 50 mm HEPES, pH 7.5, 5 mm MgCl2, 5 mm DTT, 10 ॖm ZnCl2, 0.17 (w/v) polyethylene glycol (average M r 20,000) with [3H]FPP (1–3000 nm) and Ras-CVLS protein (5–300,000 nm) as substrates as described (30Pompliano D.L. Rands E. Schaber M.D. Mosser S.D. Anthony N.A. Gibbs J.B. Biochemistry. 1992; 31: 3800-3807Crossref PubMed Scopus (249) Google Scholar). Steady-state kinetic parameters were calculated using the nonlinear analysis program k·CAT (Europa Scientific Software Corp., Hollis, NH) assuming a molecular mass of 93 kDa for FTase and 21 kDa for Ras-CVLS. The dissociation constant for FPP was measured in 50 mm HEPES, pH 7.5, 5 mmMgCl2, 5 mm DTT, 10 ॖmZnCl2, 0.27 (w/v)N-octyl-ॆ-d-glucopyranoside utilizing a SPA (31Nelson N. Anal. Biochem. 1987; 165: 287-293Crossref PubMed Scopus (36) Google Scholar, 32Hoffman R. Cammeron L. Anal. Biochem. 1992; 203: 70-75Crossref PubMed Scopus (21) Google Scholar). FTase was biotinylated using a biotinylation kit as described by the manufacturer. Biotinylated FTase (2–10 nm) was incubated with [3H]FPP (0.27–1640 nm) and 5 ॖg of neutral density streptavidin SPA beads for 30 min at room temperature after which the bound substrate was measured in a TopCount scintillation counter (Packard Instrument Co.). Scatchard plots (boundversus bound/free) of the resultant data were employed to determine the apparent K d [K d (app)] values using the program k·CAT. Zinc binding to FTase was analyzed using a modification of the method of Fu et al. (33Fu H.-W. Moomaw J.F. Moomaw C.R. Casey P.J. J. Biol. Chem. 1996; 271: 28541-28548Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). 20 ॖg of FTase in 100 ॖl of buffer A (20 mm Tris-HCl, pH 8.0, 1 mm DTT, 100 mm NaCl, and 25 ॖm EDTA) was incubated at 30 °C for 1 h and then overnight on ice to deplete the enzyme of bound Zn2+. The Zn2+-depleted FTase was buffer-exchanged into buffer B (40 mm Tris-HCl, pH 8.5, 1 mm DTT, 1 ॖm EDTA, 100 mm NaCl) using Bio-Spin-6 columns, and the protein concentration of the eluate was determined. 3.6 ॖg of FTase was brought up to 18 ॖl in buffer B, and 3 ॖl of 143 ॖm ZnCl2 (either unlabeled ZnCl2 in 0.1 n HCl or65ZnCl2) was added, and the solution was incubated at room temperature for 3 h and then on ice overnight. Two ॖl of 220 ॖm FPP was added to the samples containing labeled 65ZnCl2, and 2 ॖl of 22 ॖm [3H]FPP was added to those containing unlabeled ZnCl2. After incubating at room temperature for 15 min, the samples were electrophoresed in nondenaturing 67 polyacrylamide gels using TBE as running buffer. After electrophoresis the gels were either washed 15–30 min in TBE running buffer (65ZnCl2 samples) or soaked in ENTENSIFYTM (samples containing [3H]farnesyl diphosphate). The gels were dried between cellulose sheets and exposed to x-ray film. Alignment of the ॆ-subunits of PTases from various species identified a number of conserved amino acids (Fig. 1). Here we present the investigation of 11 conserved residues whose side chains contained aromatic rings or heteroatoms (TableI). These included residues that were shown to be near the essential Zn2+ ion in the recently published crystal structure of rat FTase (26Park J.T. Boduluri S.R. Moomaw J.F. Casey P.J. Beese L.T. Science. 1997; 275: 1800-1804Crossref PubMed Scopus (329) Google Scholar). To analyze the function of these amino acids, each was individually changed to alanine. Alanine was chosen because it has a small side chain and generally does not affect the secondary structure of a protein. E. coliexpression strains were made for these mutated FTases. All except αॆY300A were expressed at levels similar to wild type enzyme and upon purification were found to be αॆ heterodimers (data not shown). Further analysis of αॆY300A FTase suggested that this enzyme was unstable; therefore, in its place we made αॆY300F which was expressed well and was stable. We purified the 11 mutant FTases and have biochemically characterized them to elucidate the potential role(s) of these residues.Table IBiochemical properties of mutant human FTasesEnzymeK m (Ras-CVLS)K m (FPP)k CATRelativek CATK d (FPP)nm ± S.E.nm ± S.E.mol FPP/mol FTase/s ± S.E.nm ± S.E.Wild type αॆ360 ± 3011 ± 11.4 ± 0.3 × 10−213.3 ± 0.4αॆR202A170,000 ± 40,0004.1 ± 0.26.4 ± 1.3 × 10−30.463.0 ± 0.65αॆH248A570 ± 6094 ± 381.5 ± 0.3 × 10−21.119 ± 2αॆC254A470 ± 2015 ± 23.3 ± 0.6 × 10−22.46.2 ± 0.9αॆR291A2900 ± 600400 ± 301.0 ± 0.2 × 10−17.146 ± 4αॆK294A2000 ± 200410 ± 304.2 ± 0.4 × 10−2343 ± 6αॆD297A2500 ± 4005.9 ± 1.22.3 ± 0.4 × 10−40.0163.1 ± 0.5αॆC299AND1-aND, not determined.ND1.0 ± 0.2 × 10−51-bFor αॆC299A the specific activity determined using 400 nm FPP and 4000 nm Ras-CVLS is indicated.0.000931-bFor αॆC299A the specific activity determined using 400 nm FPP and 4000 nm Ras-CVLS is indicated.4.9 ± 1.3αॆY300F190 ± 307.2 ± 1.92.9 ± 0.7 × 10−30.2110 ± 1αॆW303A3800 ± 500210 ± 302.8 ± 0.4 × 10−22.017 ± 3αॆD359A2200 ± 4005.8 ± 1.71.6 ± 0.2 × 10−30.115.7 ± 0.6αॆH362A2900 ± 4008.1 ± 1.21.5 ± 0.2 × 10−30.113.0 ± 0.7The kinetic parameters were determined as described under 舠Experimental Procedures舡 (n = 3–20).1-a ND, not determined.1-b For αॆC299A the specific activity determined using 400 nm FPP and 4000 nm Ras-CVLS is indicated. Open table in a new tab The kinetic parameters were determined as described under 舠Experimental Procedures舡 (n = 3–20). Kinetic parameters (K m FPP,K m Ras-CVLS, andk CAT) and the apparent dissociation constant for FPP (FPP K d (app)) for each enzyme were determined (Table I). Catalytic activity (k CAT) of 5 of the 11 mutant human FTases was deficient relative to wild type enzyme. FTase αॆC299A was nearly inactive, whereas the catalytic activities of αॆD297A, αॆY300F, αॆD359A, and αॆH362A were 5–100-fold lower than that of wild type FTase. Catalytic activities of the other mutant FTases were similar or somewhat higher than for wild type enzyme. The crystal structure of rat FTase shows that amino acids Asp-ॆ297, Cys-ॆ299, and His-ॆ362 are ligands for the essential Zn2+ ion; thus mutations in these residues would be expected to inhibit enzyme activity (26Park J.T. Boduluri S.R. Moomaw J.F. Casey P.J. Beese L.T. Science. 1997; 275: 1800-1804Crossref PubMed Scopus (329) Google Scholar). The protein substrateK m values for αॆD297A and αॆH362A were elevated approximately 7–8-fold relative to wild type FTase, but the FPP K m and K d (app)values were similar to wild type (Table I). αॆC299A was too inactive for K m value determinations. Another catalytically deficient mutant FTase, αॆD359A, was also found to have a similarly elevated protein substrate K m and wild type FPP K m and FPPK d (app) values. Mutations of ॆ-subunit residues Arg-291, Lys-294, or Trp-303 increased the K m values for both protein and prenyl substrates and the FPP K d (app), but did not lower the catalytic activity of these enzymes (Table I). In fact all three mutant enzymes had somewhat elevatedk CAT values with the k CATvalue for αॆR291A being approximately 7-fold higher than wild type FTase. Enzymatic analysis of another mutant, αॆH248A, suggested that it may have a defect in FPP binding and K m, but a near-normal protein substrate K m. Mutant FTase αॆR202A was found to have a greatly (>400-fold) elevated K m for Ras-CVLS. The FPPK m, FPP K d (app) andk CAT values for αॆR202A were similar to wild type FTase. FTase αॆY300F had a slightly (∼5-fold) decreasedk CAT. The final mutant FTase examined, αॆC254A, showed no obvious defects in the measured parameters. FTase tightly binds a single Zn2+ ion that is essential for activity (12Reiss Y. Brown M.S. Goldstein J.L. J. Biol. Chem. 1992; 267: 6403-6408Abstract Full Text PDF PubMed Google Scholar, 13Chen W.-J. Moomaw J.F. Overton L. Kost T.A. Casey P.J. J. Biol. Chem. 1993; 268: 9675-9680Abstract Full Text PDF PubMed Google Scholar). Typical amino acid ligands for Zn2+ include Asp, Glu, His, and Cys. We tested nine of the mutant enzymes for binding to radioactive 65Zn2+ using a native gel system described previously (33Fu H.-W. Moomaw J.F. Moomaw C.R. Casey P.J. J. Biol. Chem. 1996; 271: 28541-28548Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The mutants tested included all 6 in which His, Cys, and Asp had been mutated (no Glu residues were mutated). As a control for enzyme integrity, we also examined, in parallel, [3H]FPP binding in the same gel system. The results indicated that four of the mutant FTases, αॆD297A, αॆC299A, αॆD359A, and αॆH362A, were defective in Zn2+binding but retained the ability to bind [3H]FPP (Fig.2). Three of these amino acids, Asp-ॆ297, Cys-ॆ299, and His-ॆ362, appear as ligands in the crystal structure of rat FTase (26Park J.T. Boduluri S.R. Moomaw J.F. Casey P.J. Beese L.T. Science. 1997; 275: 1800-1804Crossref PubMed Scopus (329) Google Scholar), with Cys-ॆ299 of rat FTase previously being shown to be a Zn2+ ligand by biochemical and molecular techniques (33Fu H.-W. Moomaw J.F. Moomaw C.R. Casey P.J. J. Biol. Chem. 1996; 271: 28541-28548Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Asp-ॆ359, although near the Zn2+ ion, did not appear to be a ligand in the rat FTase structure. The kinetic parameters shown in Table I were determined in the presence of 10 ॖm ZnCl2, thus mutant FTases that have a Zn2+ binding defect may have had that defect partially masked. Since the method used to purify mutant and wild type FTases involved adding excess ZnCl2 to the enzyme preparation before the final ion exchange step, enzymes that tightly bound Zn2+ should have retained this metal ion and not require added Zn2+ for activity. We, therefore, assayed the enzyme activity of wild type and several mutant FTases adding either 100 ॖm EDTA (−ZnCl2), to scavenge residual free Zn2+, or 10 ॖm ZnCl2(+ZnCl2). FTase αॆC299A was not tested since it is essentially inactive even in the presence of added ZnCl2(Table I). The catalytic rates of wild type, αॆH248A, and αॆC254A FTases were found to be independent of added ZnCl2 (Table II). The activity of mutant FTases αॆD297A, αॆD359A, and αॆH362A, however, showed 10-fold or greater dependence on exogenously added ZnCl2, with the activity in the absence of ZnCl2 being at the limit of detection (∼10−5mol of FPP incorporated/mol of FTase/s).Table IIZn2+ dependence of wild type and mutant FTase activityFTaseActivity −ZnCl2Activity +ZnCl2mol FPP/mol FTase/s ± S.E.Wild type1.0 ± 0.1 × 10−29.1 ± 1.0 × 10−3αॆH248A9.1 ± 0.5 × 10−31.1 ± 0.1 × 10−2αॆC254A2.1 ± 0.2 × 10−22.5 ± 0.3 × 10−2αॆD297A1.2 ± 0.4 × 10−51.3 ± 0.1 × 10−4αॆD359A2.2 ± 0.6 × 10−51.4 ± 0.2 × 10−3αॆH362A2.7 ± 0.4 × 10−51.4 ± 0.2 × 10−3Wild type and mutant (αॆH248A, αॆC254A, αॆD297A, αॆD359A, αॆH362A) FTases were assayed for enzymatic activity at 30 °C in 50 mm HEPES, pH 7.5, 5 mmMgCl2, 5 mm DTT, 0.17 (w/v) polyethylene glycol (average M r 20,000) plus either 100 ॖmEDTA (−ZnCl2) or 100 ॖm ZnCl2(+ZnCl2). Substrates were used at 5–10 times theK m value for [3H]FPP and atK m for Ras-CVLS. Reactions were run in triplicate, and time points were taken at 3–6 min intervals to determine the rate of [3H]FPP incorporation. Open table in a new tab Wild type and mutant (αॆH248A, αॆC254A, αॆD297A, αॆD359A, αॆH362A) FTases were assayed for enzymatic activity at 30 °C in 50 mm HEPES, pH 7.5, 5 mmMgCl2, 5 mm DTT, 0.17 (w/v) polyethylene glycol (average M r 20,000) plus either 100 ॖmEDTA (−ZnCl2) or 100 ॖm ZnCl2(+ZnCl2). Substrates were used at 5–10 times theK m value for [3H]FPP and atK m for Ras-CVLS. Reactions were run in triplicate, and time points were taken at 3–6 min intervals to determine the rate of [3H]FPP incorporation. FTase αॆR202A was found to have aK m value for Ras-CVLS >400-fold higher than that for wild type FTase with no defect in FPP K m orK d (app) (Table I). An explanation for this may be that the negatively charged carboxylate of the CAAX substrate interacts with the positively charged guanidino side chain of the arginine residue. To test this hypothesis, we looked at the ability of four CAAX-competitive FTase inhibitors (FTI) to inhibit the activity of this mutant enzyme versus wild type (Fig. 3 and Table III). The peptide FTI, CVFM, and the peptidomimetic FTI, L-739,750, both contain a cysteine thiol and a free C-terminal carboxylate. L-739,787 is similar to L-739,750 except that the C-terminal methionine sulfone has been reduced to methioninol. The FTI, L-745,631, is a tripeptide mimetic that contains a free thiol, but no carboxylate. All four inhibitors are members of FTI classes that have been shown to be competitive with the CAAX substrate, but not competitive with FPP (30Pompliano D.L. Rands E. Schaber M.D. Mosser S.D. Anthony N.A. Gibbs J.B. Biochemistry. 1992; 31: 3800-3807Crossref PubMed Scopus (249) Google Scholar, 34Williams T.M. Ciccarone T.M. MacTough S.L. Bock R.L. Conner M.W. Davide J.P. Hamilton K. Koblan K.S. Kohl N.E. Kral A.M. Mosser S.D. Omer C.A. Pompliano D.L. Rands E. Schaber M.D. Shah D. Wilson F.R. Gibbs J.B. Graham S.L. Hartman G.D. Oliff A.I. Smith R.L. J. Med. Chem. 1996; 39: 1345-1348Crossref PubMed Scopus (58) Google Scholar, 35Koblan K.S. Culberson J.C. deSolms S.J. Giuliani E.A. Mosser S.D. Omer C.A. Pitzenberger S.M. Bogusky M.J. Protein Sci. 1995; 4: 681-688Crossref PubMed Scopus (46) Google Scholar). IC50 values were determined for the four compounds under K m conditions for the respective enzymes (Table III). Under these conditions, the IC50 values should be approximately twice the K i value for a competitive inhibitor (36Cheng Y.C. Prusoff W.H. Biochem. Pharm. 1973; 22: 3099-3108Crossref PubMed Scopus (12215) Google Scholar). The two carboxylate-containing inhibitors, CVFM and L-739,750, had 100–200-fold higher IC50 values for αॆR202A as compared with wild type FTase. The IC50 value for L-739,787, in which the carboxylate has been reduced to an alcohol, was less than 4-fold higher for αॆR202A as compared with wild type FTase. This indicates, at most, a slight difference in binding of L-739,787 to the two enzymes. The IC50 values for the non-carboxylate FTI, L-745,631, were within 2-fold of one another indicating that it binds to both enzymes with similar affinity. Altogether these data are consistent with Arg-ॆ202 interacting with the free carboxylate of the CAAX competitive FTIs and by inference with the carboxylate of the CAAX substrate.Table IIIInhibition of wild type and αॆR202A FTase by farnesyl transferase inhibitorsFarnesyltransferase inhibitorIC50 wild type ± S.E.IC50 αॆR202A ± S.E.nmCVFM45 ± 911,000 ± 1,000L-739,7503.1 ± 0.5290 ± 20L-739,7871600 ± 3006200 ± 1200L-745,6311.8 ± 0.13.1 ± 0.5Reactions were performed at 30 °C using the buffer conditions described under 舠Experimental Procedures,舡 100 nm[3H]FPP, K m concentrations of Ras-CVLS, 0.5–1.0 nm enzyme, and varying amounts of the following FTIs: CVFM, L-739,750, L-745,631, and L-739,787. The IC50values were determined by fitting the data to the equation [y = 100/(1 + (x/IC50)n)] with n = slope of the curve through the IC50. Open table in a new tab Reactions were performed at 30 °C using the buffer conditions described under 舠Experimental Procedures,舡 100 nm[3H]FPP, K m concentrations of Ras-CVLS, 0.5–1.0 nm enzyme, and varying amounts of the following FTIs: CVFM, L-739,750, L-745,631, and L-739,787. The IC50values were determined by fitting the data to the equation [y = 100/(1 + (x/IC50)n)] with n = slope of the curve through the IC50. In this communication we have examined the roles of 11 conserved residues of the ॆ-subunit of human FTase. From the crystal structure of the closely related rat FTase, all of these residues are in the vicinity of the essential Zn2+ ion (26Park J.T. Boduluri S.R. Moomaw J.F. Casey P.J. Beese L.T. Science. 1997; 275: 1800-1804Crossref PubMed Scopus (329) Google Scholar). Mutations in a number of these conserved residues affected the kinetic and biochemical properties of the enzyme (Table I). Mutations in Asp-297, Cys-299, Tyr-300, Asp-359, and His-362 of the FTase ॆ-subunit decreased the catalytic efficiency 5–1000-fold. Protein substrate K m values for human FTase αॆR202A were much higher than for wild type enzyme, but this mutant showed no defect in FPP binding or utilization (Table I). FTase αॆR202A was 100–200-fold less sensitive than wild type enzyme to protein substrate competitive FTIs that contain a carboxylate while being similarly sensitive to protein substrate competitive FTIs lacking a free carboxylate (Table III). The structure of rat FTase shows that Arg-ॆ202 is near Tyr-ॆ361 and that both are in the vicinity of the Zn2+ ion (26Park J.T. Boduluri S.R. Moomaw J.F. Casey P.J. Beese L.T. Science. 1997; 275: 1800-1804Crossref PubMed Scopus (329) Google Scholar). Amino acid changes of Tyr-ॆ362 of yeast FTase, which is homologous to Tyr-ॆ361 of rat and human FTase, affects the X residue specificity of the CAAXsubstrate (37Del Villar K. Mitsuzawa H. Yang W. Sattler I. Tamanoi F. J. Biol. Chem. 1997; 272: 680-687Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The X residue of the protein substrate is the C-terminal amino acid containing a free carboxylate. Altogether, these data strongly suggest that Arg-ॆ202 interacts with the C-terminal carboxylate of the X residue of CAAXsubstrates. The interaction of the negatively charged carboxylate could be with the positively charged guanidino group of the arginine side chain. Given the difference in relative IC50 values for L-739,787 (C-terminal alcohol) and L-739,750 (C-terminal carboxylate) for wild type and αॆR202A, this is probably through an ionic interaction. Three mutant FTases, αॆR291A, αॆK294A, and αॆW303A, had elevated FPP and protein substrate K m values. The elevated FPP K d (app) for αॆR291A, αॆK294A, and αॆW303A indicate that these three mutant FTases have a primary FPP binding defect (Table I). The evidence for an FPP binding defect for αॆW303A was less convincing as theK d (app) value was only 5-fold higher than wild type. Since FPP binds before the protein substrate (10Pompliano D.L. Schaber M.D. Mosser S.D. Omer C.A. Shafer J.A. Gibbs J.B. Biochemistry. 1993; 32: 8341-8347Crossref PubMed Scopus (109) Google Scholar, 11Furfine E.S. Leban J.J. Landavazo A. Moomaw J.F. Casey P.J. Biochemistry. 1995; 34: 6857-6862Crossref PubMed Scopus (161) Google Scholar), it may be that the elevated protein substrate K m values for these three mutant FTases are caused by the defect in FPP binding. In the crystal structure of rat FTase, Trp-ॆ303 is in a hydrophobic pocket that was proposed to be where the farnesyl chain binds (26Park J.T. Boduluri S.R. Moomaw J.F. Casey P.J. Beese L.T. Science. 1997; 275: 1800-1804Crossref PubMed Scopus (329) Google Scholar). Arg-ॆ291 and Lys-ॆ294 are near the surface of this pocket. In the rat FTase structure paper it was postulated that Arg-ॆ202 might be the amino acid that interacts with the negatively charged phosphates of FPP. We believe, however, that our data with these mutant FTases indicate that Arg-ॆ291 and Lys-ॆ294 are likely to be residues that interact with these phosphates and that Arg-ॆ202 interacts with the CAAX carboxylate. An additional mutant FTase that had a defect in FPP interaction (K d (app) and K m) was αॆH248A (Table I). His-ॆ248 is a residue near the Zn2+ ion and nearby Arg-ॆ291 and Lys-ॆ294 in rat FTase (26Park J.T. Boduluri S.R. Moomaw J.F. Casey P.J. Beese L.T. Science. 1997; 275: 1800-1804Crossref PubMed Scopus (329) Google Scholar). The crystal structure of rat FTase identified Asp-ॆ297, Cys-ॆ299, and His-ॆ362 as Zn2+ ligands (26Park J.T. Boduluri S.R. Moomaw J.F. Casey P.J. Beese L.T. Science. 1997; 275: 1800-1804Crossref PubMed Scopus (329) Google Scholar). ॆC299A has also been shown to be a Zn2+ ligand by biochemical and molecular techniques (33Fu H.-W. Moomaw J.F. Moomaw C.R. Casey P.J. J. Biol. Chem. 1996; 271: 28541-28548Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Changing any one of these three residues or Asp-ॆ359 to alanine inhibited binding of 65Zn2+ (Fig.2). All four mutant enzymes bound FPP withK d (app) values similar to wild type FTase (Table I). The ability of these mutants to bind FPP is consistent with previous results indicating that isoprenoid binding is Zn2+-independent (12Reiss Y. Brown M.S. Goldstein J.L. J. Biol. Chem. 1992; 267: 6403-6408Abstract Full Text PDF PubMed Google Scholar). The residual catalytic activity of αॆD297A, αॆD359A, and αॆH362A (αॆC299A was essentially inactive) was shown to be dependent on added Zn2+ (TableII). While in the rat FTase crystal structure Asp-ॆ359 is too far away from the Zn2+ ion to be a ligand, these data suggest that it may have an effect on Zn2+ binding. Whether the effect is direct or indirect is presently unclear. Besides mutations in residues that affected Zn2+ ion binding, the only other mutant FTase that decreasedk CAT was αॆY300F (Table I). Thek CAT of this enzyme was reduced about 5-fold relative to wild type enzyme. In the crystal structure of rat FTase, Tyr-ॆ300 appears to be close to the Zn2+ ion (26Park J.T. Boduluri S.R. Moomaw J.F. Casey P.J. Beese L.T. Science. 1997; 275: 1800-1804Crossref PubMed Scopus (329) Google Scholar). While mutation of this tyrosine to phenylalanine may simply cause a localized effect on the position of the two substrates, it may also indicate a catalytic role for this residue. In glutathioneS-transferases the hydroxyl of a conserved tyrosine residue has been shown both structurally and biochemically to stabilize the catalytic thiolate anion (38Liu S. Zhang P. Ji X. Johnson W.W. Gilliland G.L. Armstrong R.N. J. Biol. Chem. 1992; 267: 4296-4299Abstract Full Text PDF PubMed Google Scholar, 39Ji X. Zhang P. Armstrong R.N. Gilliland G.L. Biochemistry. 1992; 31: 10169-10184Crossref PubMed Scopus (376) Google Scholar). We are currently testing whether the proximity of Tyr-ॆ300 to the Zn2+ indicates a similar role for this tyrosine in FTase. The experiments presented here shed light on the roles of 11 conserved amino acids of the ॆ-subunit of FTase in substrate binding, catalysis, and zinc binding. Additional studies will be needed to further define enzyme-substrate interactions and substrate specificity. While it appears that the essential Zn2+ ion is involved in catalysis, it is not clear exactly how this occurs or what other contributions the enzyme might make to catalysis. Further structural, molecular, and biochemical studies should permit the answering of these questions. We thank Erwin Lin for help in purifying some of the mutant FTases used in this study, Jay Gibbs for comments on the manuscript, and Allen Oliff for his continuing support of our work.