JBC270 - THE JOURNAL or BIOLOGICAL CHEMISTRY © 1995 by The...

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Unformatted text preview: THE JOURNAL or BIOLOGICAL CHEMISTRY © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 270, No. 32, Issue of August 11, pp, 19085—19040, 1995 Printed in U.S.A. Photoreactive Analogues of Prenyl Diphosphates as Inhibitors and Probes of Human Protein Farnesyltransferase and Geranylgeranyltransferase Type 1* (Received for publication, April 7, 1995, and in revised form, May 26, 1995) Yuri E. Bukhtiyarovili, Charles A. Omer§, and Charles M. Allenrt‘n From the iDepartment of Biochemistry and Molecular Biology, J. Hillis Miller Health Center, University of Florida, Gainesville, Florida 32610 and the §Department of Cancer Research, Merck Research Labs, West Point, Pennsylvania 19486 Photoreactive analogues of prenyl diphosphates have been useful in studying prenyltransferases. The effec- tiveness of analogues with different chain lengths as probes of recombinant human protein prenyltrans- ferases is established here. A putative geranylgeranyl diphosphate analogue, 2-diazo-3,3,3-trifluoropropiony- loxy-farnesyl diphosphate (DATFP-FPP), was the best inhibitor of both protein farnesyltransferase (PFT) and protein geranylgeranyltransferase-I (PGGT-I). Shorter photoreactive isoprenyl diphosphate analogues with ge- ranyl and dimethylallyl moieties and the DATFP-deriv- ative of famesyl monophosphate were much poorer in- hibitors. DATFP-FPP was a competitive inhibitor of both PFT and PGGT-I with K,- values of 100 and 18 nM, respectively. [32P]DATFP-FPP specifically photoradio- labeled the B-subunits of both PFT and PGGT-I. Photo- radiolabeling of PGGT-I was inhibited more effectively by geranylgeranyl diphosphate than farnesyl diphos- phate, whereas photoradiolabeling of PFT was inhibited better by famesyl diphosphate than geranylgeranyl diphosphate. These results lead to the conclusions that DATFP-FPP is an effective probe of the prenyl diphos- phate binding domains of PFT and PGGT-I. Further- more, the fl-subunits of protein prenyltransferases must contribute significantly to the recognition and binding of the isoprenoid substrate. Prenylated proteins have an important role in cellular reg- ulation, therefore, a description of their structure/function re- lationships and post-translational processing has been of great interest (1—3). Among the post-translational events is the mod- ification of cysteine residues in carboxyl termini with either a farnesyl or geranylgeranyl moiety by sequence specific protein prenyltransferases. Protein farnesyltransferase (PFT)1 is a a3- heterodimer (4, 5), which transfers the 015 isoprenoid from farnesyl diphosphate (FPP) to a Cys residue in the sequence * This work was supported in part by Grant F93UF—2 from the American Cancer Society, Florida Division, and The Cancer Center, University of Florida. 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. ll To whom correspondence should be addressed. 1 The abbreviations used are: PFT, protein farnesyltransferase; Bt, biotinylated; DATFP, diazotrifluoropropionyloxy; GPP, geranyl diphos- phate; DMAPP, dimethylallyl diphosphate; FMP, farnesyl monophos- phate; FPP, famesyl diphosphate; GGPP, geranylgeranyl diphosphate; PGGT-I, protein geranylgeranyltransferase type I; NOESY, nuclear Overhauser effect spectroscopy; DQF-COSY, double quantum filtered correlation spectroscopy; MALDI-MS, matrix assisted laser desorption ionization mass spectrometry; HPLC, high performance liquid chroma- tography; Tricine, N—[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. -Cys-A1-A2—Ser(Met, Gln), where A1 and A2 represent aliphatic amino acids (1-3, 6, 7). Protein geranylgeranyltransferase-I (PGGT-I) is also a afl-heterodimer (4), but it transfers the C20 moiety from geranylgeranyl diphosphate (GGPP) to a Cys res- idue in proteins terminating in Leu (-Cys-A1-A2-Leu) (8—11). A different class of geranylgeranyltransferases, PGGT-II or tab prenyltransferases (12), modify proteins ending in -Cys-Cys, -Cys-X-Cys, and -Cys-Cys-X-X (8, 13—15). Although, the substrate specificity of prenyltransferases which recognize the -C-A1-A2—X motif has been well established and many inhibitors of these enzymes have been described (16—20), the full extent of involvement of the protein subunits or their respective amino acid residues in the prenylation re- action has not yet been elucidated for any of these prenyltrans- ferases. A detailed description of the “active sites” of these enzymes would aid in the development of more specific enzyme inhibitors based on rational design. One means of investigating the structure of a substrate binding domain is to probe it with photoreactive substrate analogues. Earlier studies have shown photoinhibition and photolabeling of bacterial prenyltrans- ferases with a photoreactive analogue of FPP, diazotrifluoro- propionyloxy-geranyl diphosphate, DATFP-GPP (I, n = 1), (21, 22). DATFP-GPP was subsequently shown to be an inhibitor of protein prenyltransferase activity in cytosolic extracts of cul- tured human lymphocytes (23). DATFP—GPP and the GGPP analogue, DATFP-FPP (I, n = 2), have now been used to probe protein prenyltransferase function. Since PFT and PGGT-I show specificity toward both the protein and prenyl diphosphate substrates, the common a-sub- unit is not likely to be the sole determinant for substrate specificity. Accordingly, Omer et al. (5) showed that [3H]DATFP-GPP, the radiolabeled putative FPP analogue, spe- cifically labeled the B-subunit of recombinant human PFT (hPFT). Recently Yokoyama et al. (24) have synthesized [3H]DATFP-FPP and also shown that it photoradiolabeled the B-subunit of bovine brain PGGT—I. Although, the radioactive photoreactive prenyl diphosphate probes labeled only the fi-subunits of these prenyltransferases and the labeling could be inhibited by the natural substrate for each enzyme, no evidence was presented to unequivocally demonstrate that the photoprobe binds to the active site. More specifically, demon- stration that these probes inhibit the prenyltransferases com- petitively, with K,- values similar to the Km of the natural substrates, was not established. In addition, neither demon- stration of chain length specificity of the photoprobes for inhi- bition of activity was established nor was the specificity of the prenyl diphosphates in their protection of the labeling of the B-subunits determined. We present here an improved synthesis and characterization of DATFP-FPP. DATFP-FPP was shown to competitively in- 19035 19036 0 l . _ CF . (211243 [Re fl/ 3 ,Y=P207 d N2 0 RO-——Cl-I2 \ (HZ \ 0y 0 C H n II:R=H,Y:J'\ n22 CH2C1’ STRUCTURE 1. hibit both hPFT and hPGGT-I and demonstrated better inhi- bition than the shorter DATFP-isoprenyl homologues. Further- more, whereas [32P]DATFP-FPP (Structure 1) photolabeled the B-subunits of both human recombinant enzymes, inhibition of radiolabeling showed preference for the enzyme’s prenyl diphosphate substrate. EXPERIMENTAL PROCEDURES Materials—E,E-[3H]FPP (15—20 Ci/mmol) and E,E,E-[1-3H]GGPP (15—20 Ci/mmol) were purchased from American Radiolabeled Chemi- cals and DuPont NEN. Biotinylated (Bt) KTKCVIS was prepared by the Protein Chemistry Core Facility, Interdisciplinary Center for Biotech- nology Research, University of Florida. Bt-KKFFCAIL was generously provided by Dr. Alison Joly, University of California Los Angeles. FPP and GGPP were prepared as described previously (25). DATFP-GPP, [3H]DATFP-GPP (43 mCi/mmol), and DATFP-dimethylallyl diphos- phate (DATFP-DMAPP) were prepared previously (21, 22). All other reagents were purchased from Sigma unless otherwise indicated. Re- combinant hPFT, having a truncated a'subunit (hPFTaMmaB), and hPGGT-I were prepared as described previously (5, 26). Synthesis of DATFP-FPP and DATFP-FMP—The chemical synthesis of the photoreactive analogue, DATFP-FPP (I, n = 2) was accomplished by modifying the procedures described earlier (21). A similar synthesis has recently been accomplished by Yokoyama et al. (24). The principal feature of these syntheses was the oxidation of the monochloroacetyl ester of farnesol to the w-hydroxyprenyl chloroacetate. The esterifica— tion of this dihydroxy monoester with diazotrifluoropropionyl chloride gave the w—DATFP—farnesyl chloroacetate. Then selective removal of the chloroacetyl group to give DATFP-farnesol was achieved with methanolic ammonia and phosphorylation gave the final product, DATFP-FPP. A critical step for this scheme is the selective oxidation of (E,E)— 3,7,11-trimethy1-1-(chloroacetoxy)—2,6,10-dodecatriene (farnesyl chloro- acetate) to (E,E)—3,7,11-trimethyl-1-(chloroacetoxy)-2,6,10-dodecatrien- 12-01 (m-hydroxyfarnesyl chloroacetate, II). Farnesyl chloroacetate, in contrast to geranyl chloroacetate (21) was more reactive with selenious acid/t—butyl hydroperoxide. As a result, the oxidation of trans ro-termi- nal methyl group of the farnesyl group was accompanied by the oxida- tion of methylene groups as well. Increasing the extent of the reaction only led to the formation of the over-oxidation products with more than one hydroxy or carbonyl group in the molecule. Therefore, the condi— tions were optimized for the oxidation by limiting consumption of the starting material and minimizing the formation of over-oxidation prod- ucts. This was achieved by treatment for a shorter period of time and at a lower temperature than previously reported (21, 27). Farnesyl chlo- roacetate (4.6 g, 15.5 mmol) was oxidized with t-butyl hydroperoxide (3.45 ml, 31 mmol) and H2Se03 (1 g, 7.8 mmol) in 25 ml of CH2012 for 2 h at 0 EC. The w-hydroxyfarnesyl chloroacetate was obtained in 24% yield (1.18 g, 3.8 mmol) after purification by silica gel chromatography with stepwise gradient elution from petroleum ether/benzene (3:2, v/v) to benzene/ethyl acetate (35:65, v/v). The alcohol eluted with 5—10% ethyl acetate in benzene. Its R F was 0.32 in benzene/ethyl acetate (9:1, v/v). This yield represents a 6-fold increase over the other reported synthesis (24). Limited consumption of the starting material also per- mitted its recovery for subsequent utilization. Standard one—pulse DQFCOSY and NOESY experiments using a Varian Unity 600 nmr system were carried out to establish that pre- sumed structure (II) was correct. 1H nmr shifts 8 (ppm) (CZHCla) (600 MHz) Were: 1.59 (3H, s), 1.65 (3H, s), 1.71 (3H, s), 1.98 (2H, tr), 2.08 (2H, tr), 2.09 (4H, overlapping triplets), 3.96 (2H, s), 4.04 (2H, s), 4.69 (2H, d), 5.08 (1H, tr), 5.34 (1H, tr), 5.36 (1H, tr). The DQFCOSY and NOESY analyses show that monohydroxylation of the terminal trans-methyl group was achieved. The presence of three methyl peaks and four methylene signals in the spectrum rule out the possibility that II was a secondary alcohol. The alcoholic group obviously resides on the terminal carbon because the symmetrized DQFCOSY spectrum showed that the methylene protons a (5, 3.9 ppm), assigned to the oxidized carbon, and the vinyl proton c (5, 5.36 ppm) were scalar coupled. The a protons were Photoaffinity Labeling of Prenyltransferases coupled long range to the methylene protons d (6, 2.09 ppm) and the methyl group b (8, 1.65 ppm). The trans-orientation of the w-hydroxyl moiety was expected by analogy to the work of others, who have estab— lished that farnesylacetate (28) and other gem-dimethyl allylic com- pounds (29) are oxidized by selenium dioxide with the formation of the trans-hydroxy derivatives. This result was confirmed here by noting that the chemical shifts of the vinyl proton c (8, 5.36) and the terminal methyl group b (5, 1.65) are the values expected for a trans-allylic alcohol and are in contrast to the shifts (5, 5.25 (vinyl) and 6, 1.78 (methyl)) expected for the corresponding cis alcohol (30). Other intermediates of the synthetic pathway were pure as assessed by TLC using Kieselgel 60 F254 plates (E. Merck) and were analyzed by 1H nmr analysis using a Varian EM-390 (90 MHz) spectrometer. Their proton nmr spectral results were consistent with expected products. DATFP-farnesol was phosphorylated by a previously described method (31). The products of phosphorylation, DATFP—FMP and DATFP-FPP, were separated by DE52 cellulose (Whatman) column chromatography with a linear gradient from 25 to 500 mM ammonium acetate in 50% (v/V) aqueous methanol. DATFP-FPP was isolated in 10% yield (0.02 mmol) after further purification by chromatography on CF-ll cellulose in tetrahydrofuran, 100 mM NH4HCO3 (85:15, v/v) (25). RF = 0.30 in 2-propanol/NH4OH/HZO (6:3:1, v/v/v). 6238 = 11,600 L-mol_1~r:m_1 (in 1 mM NH4OH). Fractions containing DATFP-FMP were lyophilized and applied to an Amberlite XAD-2 column in 1 mM aqueous ammonia. The pure monophosphate was eluted with 90% methanol. RF : 0.61 in 2-propa- nol/NH4OH/H20 (623:1, v/v/v). 6240 = 9,700 L‘mol’l-cm’1 (in 1 mM NHAOH). Synthesis ofFZP]DATFP-FPP—DATFP-farnesol was also converted to its [32P]diphosphate derivative by reaction with [32P]bis-(triethylam- monium)hydrogen phosphate in the presence of a large excess of CClSCN. In a typical procedure, 2—5 mCi of carrier free H332PO4 were mixed with aqueous H3PO4 to achieve the desired specific activity (40—500 mCi/mmol). The solution was lyophilized over P205 and the calculated amount of Et3N in dry acetonitrile was added to prepare [32P]bis-(triethylammonium)hydrogen phosphate. DATFP-farnesol (50 mM) in a 20—50% solution of CCISCN in acetonitrile was added to achieve a 2 molar excess of inorganic phosphate over DATFP-farnesol. The reaction was allowed to proceed at room temperature under a blanket of inert gas for 1—2 h. The solvent and the excess CClaCN were removed in a stream of argon, and the residue was taken up with the buffer for ion~exchange chromatography (20 mM ammonium acetate in 50% MeOH). The sample was loaded onto a 1 X 17-cm column of Whatman DE52 cellulose equilibrated with the same buffer. After washing the column with the buffer, radioactive mono- and diphos- phates of DATFP-farnesol were eluted consecutively with 100 mM am- monium acetate in 50% MeOH. Fractions were analyzed by TLC. Those containing [32P]DATFP-FPP were combined, evaporated, and lyophi- lized. In some cases the product was further purified by chromatogra— phy on Kieselgel 60 plates (250 um, E. Merck) in 2-propanol/NH4OH/ H20 (6:3:1, v/v/v). Radiochemical purity of the product was at least 70% as assessed by thin‘layer radiochromatography. Protein Prenyltransferase Assay—hPFT and hPGGT—I were assayed with Bt—peptide and the appropriate [3H]prenyl diphosphate (32, 33). hPFT incubation mixtures (100 pl) contained 69 mM potassium phos- phate buffer, pH 7.0, 55 uM ZnClZ, 5.5 mM MgClz, and 10 mM dithio- threitol. hPGGT-I incubation mixtures (180—200 ul) were modified after those described by Zhang et al. (34) with the addition of 0.2% polyvinyl alcohol (5). The specific concentrations of Bt-KTKCVIS, Bt— KKFFCAIL, [3H]FPP, and [3H]GGPP and enzyme are given in the legends to the figures. Control assay mixtures omitted the biotinylated peptide. Incubations were carried out at 37 °C for 30 min followed by the addition of a suspension of 0.36—0.61 units of avidin-agarose beads in a solution containing 25 mM EDTA, 0.5 M NaCl, and either 2 mM FPP or GGPP for the hPFT and hPGGT—I assays, respectively. After incu- bation for another 10 min at 37 °C, the beads were washed as described previously (11), collected analytically on GN-6 Metricel (Gelman) mem— brane filters, and analyzed for radioactivity. Inhibition Experiments-#Photoprobes to be tested as inhibitors were added from stock solutions (made basic with aqueous ammonia) to reaction mixtures containing the enzyme with either [3H]FPP or [3H] GGPP and the appropriate Bt-peptide. Prenyltransferase assays were carried out in the dark. Km values were determined from the intercept (—1/S) of Lineweaver-Burke plots at UV 2 0. K, value was estimated from the slopes of the double reciprocal plots in the presence of inhib- itor. All kinetic analyses were conducted with saturating levels of the appropriate Bt-peptide. The data reported in Table I and all figures represent the average of duplicate determinations. The standard error Photoaffinity Labeling of Prenyltransferases of the enzymatic activity determinations did not exceed 10%. The mean values for two repeats were taken for calculation of kinetic parameters using the Enzfitter program (44). Photolabeling Experiments—Recombinant hPFT or hPGGT-I (2—7 ug) were pre-equilibrated with [32PJDATFP-FPP for 5 min at room temperature in 25 mM potassium phosphate buffer, pH 7.0, containing 2 mM MgCl2 and 20 MM ZnCl2 in a total volume of 30—60 I~'~1- Open microcentrifuge tubes containing the samples were placed under a bactericidal lamp, cooled to 4 °C, and irradiated for 6—10 min. Electro- phoresis sample buffer was then added to each tube and the samples were analyzed by SDS-polyacrylamide gel electrophoresis on a 10% Tris-Tricine gel (35). The gel was silver stained (36), dried, and auto- radiographed using Fuji RX x-ray film. In some cases the radioactive bands corresponding to the a- and B-subunits were excised, transferred to scintillation vials, and counted for radioactivity. HPLC and MALDI—MS Analyses—Incubation mixtures for HPLC analysis of hPFT reaction products contained in a final volume of 410 p.12 6.1 mM potassium phosphate buffer, pH 7.0, 0.5 mM MgC12, 5 ,uM ZnClZ, 9.8 mM dithiothreitol, 61 uM KTKCVIS, 122 uM allylic diphos- phate, and 29 pmol of recombinant hPFT. Reactions proceeded at 37 °C for up to 6 h. At different times, the reaction was stopped by freezing the samples at —18 °C. Aliquots (50 al) from these samples were then analyzed by HPLC using a Perkin-Elmer Series 4 liquid chromatograph equipped with a Perkin-Elmer analytical C18 column (4.6 X 250 mm) and an LC-75 spectrophotometric detector preset to 215 nm. Mixtures of solvent A (0.1% trifluoroacetic acid in water) and solvent B (0.1% trifluoroacetic acid in acetonitrile) were used for gradient elution. The column was equilibrated for 1 min with 2% B and then eluted with a concave gradient to 60% B (19 min) followed by a linear gradient to 80% B (10 min). Matrix-assisted Laser Desorption Ionization Mass Spectrometry (MALDLMS) of the isolated farnesylated peptide was performed on a Voyager RP MALDI time-of-flight mass spectrometer (PerSeptive Bio- systems) in a positive ion analysis mode using a—cyano-4-hydroxycin- namic acid as a matrix. RESULTS Inhibition of Recombinant Human PFT and PGGT-I by Various DATFPvDerivatives—Four DATFP-derivatives were tested as inhibitors of hPFT and hPGGT-I. DATFP-FPP showed distinctly better inhibition of hPFT than the shorter chain analogues (DATFP-GPP and DATFP-DMAPP) (Fig. 1A). The monophosphate of DATFP-farnesol (DATFP-FMP) was a much poorer inhibitor than the corresponding diphosphate with inhibitory properties similar to DATFP-GPP. The effec- tiveness of these inhibitors on hPGGT-I was similar to that exhibited with hPFT, except that DATFP-GPP was no better an inhibitor than the shorter analogue DATFP—DMAPP (Fig. 1B). Inhibition of Recombinant hPFT and hPGGT—I by DATFP- FPP—Double reciprocal plots of the activities of hPFT in the presence of two different concentrations of DATFP-FPP showed competitive inhibition (Fig. 2A) with a K,- of 100 nM. This X,- value was only slightly higher than the estimated Km of 20 nM for FPP, which was determined in the same experiment. Sim- ilarly, hPGGT-I was competitively inhibited by DATFP-FPP with a K value of 18 nM, whereas the estimated K"1 value of 16 nM was observed for its isoprenoid substrate, GGPP (Fig. 28). The inhibition kinetics could also be a manifestation of the function of the DATFP-derivatives as alternative substrates. Product formation from the reaction of the DATFP—derivatives and peptide with hPFT was assessed by two independent meth- ods. No detectable product appearance (or loss ...
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