©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Photoreactive Analogues of Prenyl Diphosphates as Inhibitors and Probes of Human Protein Farnesyltransferase and Geranylgeranyltransferase Type I (*)

(Received for publication, April 7, 1995; and in revised form, May 26, 1995 )

Yuri E. Bukhtiyarov (1) Charles A. Omer (2) Charles M. Allen (1)(§)

From the (1)Department of Biochemistry and Molecular Biology, J. Hillis Miller Health Center, University of Florida, Gainesville, Florida 32610 and the (2)Department of Cancer Research, Merck Research Labs., West Point, Pennsylvania 19486

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Photoreactive analogues of prenyl diphosphates have been useful in studying prenyltransferases. The effectiveness of analogues with different chain lengths as probes of recombinant human protein prenyltransferases is established here. A putative geranylgeranyl diphosphate analogue, 2-diazo-3,3,3-trifluoropropionyloxy-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 geranyl and dimethylallyl moieties and the DATFP-derivative of farnesyl monophosphate were much poorer inhibitors. DATFP-FPP was a competitive inhibitor of both PFT and PGGT-I with K values of 100 and 18 nM, respectively. [P]DATFP-FPP specifically photoradiolabeled the beta-subunits of both PFT and PGGT-I. Photoradiolabeling of PGGT-I was inhibited more effectively by geranylgeranyl diphosphate than farnesyl diphosphate, whereas photoradiolabeling of PFT was inhibited better by farnesyl diphosphate than geranylgeranyl diphosphate. These results lead to the conclusions that DATFP-FPP is an effective probe of the prenyl diphosphate binding domains of PFT and PGGT-I. Furthermore, the beta-subunits of protein prenyltransferases must contribute significantly to the recognition and binding of the isoprenoid substrate.


INTRODUCTION

Prenylated proteins have an important role in cellular regulation, therefore, a description of their structure/function relationships and post-translational processing has been of great interest(1, 2, 3) . Among the post-translational events is the modification of cysteine residues in carboxyl termini with either a farnesyl or geranylgeranyl moiety by sequence specific protein prenyltransferases. Protein farnesyltransferase (PFT) (^1)is a alphabeta-heterodimer(4, 5) , which transfers the C isoprenoid from farnesyl diphosphate (FPP) to a Cys residue in the sequence -Cys-A(1)-A(2)-Ser(Met, Gln), where A(1) and A(2) represent aliphatic amino acids (1, 2, 3, 6, 7) . Protein geranylgeranyltransferase-I (PGGT-I) is also a alphabeta-heterodimer(4) , but it transfers the C moiety from geranylgeranyl diphosphate (GGPP) to a Cys residue in proteins terminating in Leu (-Cys-A(1)-A(2)-Leu)(8, 9, 10, 11) . A different class of geranylgeranyltransferases, PGGT-II or rab prenyltransferases(12) , modify proteins ending in -Cys-Cys, -Cys-X-Cys, and -Cys-Cys-X-X(8, 13, 14, 15) .

Although, the substrate specificity of prenyltransferases which recognize the -C-A(1)-A(2)-X motif has been well established and many inhibitors of these enzymes have been described(16, 17, 18, 19, 20) , the full extent of involvement of the protein subunits or their respective amino acid residues in the prenylation reaction has not yet been elucidated for any of these prenyltransferases. 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 prenyltransferases with a photoreactive analogue of FPP, diazotrifluoropropionyloxy-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 cultured 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 alpha-subunit 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, specifically labeled the beta-subunit of recombinant human PFT (hPFT). Recently Yokoyama et al.(24) have synthesized [^3H]DATFP-FPP and also shown that it photoradiolabeled the beta-subunit of bovine brain PGGT-I. Although, the radioactive photoreactive prenyl diphosphate probes labeled only the beta-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, demonstration that these probes inhibit the prenyltransferases competitively, with K values similar to the K of the natural substrates, was not established. In addition, neither demonstration of chain length specificity of the photoprobes for inhibition of activity was established nor was the specificity of the prenyl diphosphates in their protection of the labeling of the beta-subunits determined.

We present here an improved synthesis and characterization of DATFP-FPP. DATFP-FPP was shown to competitively inhibit both hPFT and hPGGT-I and demonstrated better inhibition than the shorter DATFP-isoprenyl homologues. Furthermore, whereas [P]DATFP-FPP (Fig.Z1) photolabeled the beta-subunits of both human recombinant enzymes, inhibition of radiolabeling showed preference for the enzyme's prenyl diphosphate substrate.


Figure Z1: Structure 1




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 Chemicals and DuPont NEN. Biotinylated (Bt) KTKCVIS was prepared by the Protein Chemistry Core Facility, Interdisciplinary Center for Biotechnology 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 diphosphate (DATFP-DMAPP) were prepared previously(21, 22) . All other reagents were purchased from Sigma unless otherwise indicated. Recombinant hPFT, having a truncated alpha-subunit (hPFTalpha), 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 -hydroxyprenyl chloroacetate. The esterification of this dihydroxy monoester with diazotrifluoropropionyl chloride gave the -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-trimethyl-1-(chloroacetoxy)-2,6,10-dodecatriene (farnesyl chloroacetate) to (E,E)-3,7,11-trimethyl-1-(chloroacetoxy)-2,6,10-dodecatrien-12-ol (-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 -terminal methyl group of the farnesyl group was accompanied by the oxidation 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 conditions were optimized for the oxidation by limiting consumption of the starting material and minimizing the formation of over-oxidation products. This was achieved by treatment for a shorter period of time and at a lower temperature than previously reported(21, 27) . Farnesyl chloroacetate (4.6 g, 15.5 mmol) was oxidized with t-butyl hydroperoxide (3.45 ml, 31 mmol) and H(2)SeO(3) (1 g, 7.8 mmol) in 25 ml of CH(2)Cl(2) for 2 h at 0 °C. The -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 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 permitted 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 presumed structure (II) was correct. ^1H nmr shifts (ppm) (C^2HCl(3)) (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 (, 3.9 ppm), assigned to the oxidized carbon, and the vinyl proton c (, 5.36 ppm) were scalar coupled. The a protons were coupled long range to the methylene protons d (, 2.09 ppm) and the methyl group b (, 1.65 ppm). The trans-orientation of the -hydroxyl moiety was expected by analogy to the work of others, who have established that farnesylacetate (28) and other gem-dimethyl allylic compounds (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 (, 5.36) and the terminal methyl group b (, 1.65) are the values expected for a trans-allylic alcohol and are in contrast to the shifts (, 5.25 (vinyl) and , 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 F 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-11 cellulose in tetrahydrofuran, 100 mM NH(4)HCO(3) (85:15, v/v)(25) . R = 0.30 in 2-propanol/NH(4)OH/H(2)O (6:3:1, v/v/v). = 11,600 Lbulletmolbulletcm (in 1 mM NH(4)OH).

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. R = 0.61 in 2-propanol/NH(4)OH/H(2)O (6:3:1, v/v/v). = 9,700 Lbulletmolbulletcm (in 1 mM NH(4)OH).

Synthesis of [P]DATFP-FPP

DATFP-farnesol was also converted to its [P]diphosphate derivative by reaction with [P]bis-(triethylammonium)hydrogen phosphate in the presence of a large excess of CCl(3)CN. In a typical procedure, 2-5 mCi of carrier free H(3)PO(4) were mixed with aqueous H(3)PO(4) to achieve the desired specific activity (40-500 mCi/mmol). The solution was lyophilized over P(2)O(5) and the calculated amount of Et(3)N in dry acetonitrile was added to prepare [P]bis-(triethylammonium)hydrogen phosphate. DATFP-farnesol (50 mM) in a 20-50% solution of CCl(3)CN 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 CCl(3)CN 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 17-cm column of Whatman DE52 cellulose equilibrated with the same buffer. After washing the column with the buffer, radioactive mono- and diphosphates of DATFP-farnesol were eluted consecutively with 100 mM ammonium acetate in 50% MeOH. Fractions were analyzed by TLC. Those containing [P]DATFP-FPP were combined, evaporated, and lyophilized. In some cases the product was further purified by chromatography on Kieselgel 60 plates (250 µm, E. Merck) in 2-propanol/NH(4)OH/H(2)O (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 µl) contained 69 mM potassium phosphate buffer, pH 7.0, 55 µM ZnCl(2), 5.5 mM MgCl(2), and 10 mM dithiothreitol. hPGGT-I incubation mixtures (180-200 µl) 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 incubation for another 10 min at 37 °C, the beads were washed as described previously(11) , collected analytically on GN-6 Metricel (Gelman) membrane 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. K values were determined from the intercept (-1/S) of Lineweaver-Burke plots at 1/V = 0. Kvalue was estimated from the slopes of the double reciprocal plots in the presence of inhibitor. All kinetic analyses were conducted with saturating levels of the appropriate Bt-peptide. The data reported in Table1and all figures represent the average of duplicate determinations. The standard error 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 µg) were pre-equilibrated with [P]DATFP-FPP for 5 min at room temperature in 25 mM potassium phosphate buffer, pH 7.0, containing 2 mM MgCl(2) and 20 µM ZnCl(2) in a total volume of 30-60 µl. Open microcentrifuge tubes containing the samples were placed under a bactericidal lamp, cooled to 4 °C, and irradiated for 6-10 min. Electrophoresis 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 autoradiographed using Fuji RX x-ray film. In some cases the radioactive bands corresponding to the alpha- and beta-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 µl: 6.1 mM potassium phosphate buffer, pH 7.0, 0.5 mM MgCl(2), 5 µM ZnCl(2), 9.8 mM dithiothreitol, 61 µM KTKCVIS, 122 µM allylic diphosphate, 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 µl) from these samples were then analyzed by HPLC using a Perkin-Elmer Series 4 liquid chromatograph equipped with a Perkin-Elmer analytical C(18) column (4.6 250 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 (MALDI-MS) of the isolated farnesylated peptide was performed on a Voyager RP MALDI time-of-flight mass spectrometer (PerSeptive Biosystems) in a positive ion analysis mode using alpha-cyano-4-hydroxycinnamic acid as a matrix.


RESULTS

Inhibition of Recombinant Human PFT and PGGT-I by Various DATFP-Derivatives

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 effectiveness 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).


Figure 1: Inhibition of hPFT and hPGGT-I by various DATFP-prenyl diphosphates. Assays were carried out as described under ``Experimental Procedures'' using (A) hPFT, 2 ng/ml, with 50 nM [^3H]FPP and 10 µM Bt-KTKCVIS or (B) hPGGT-I, 1 ng/ml, with 45 nM [^3H]GGPP, 1.5 µM Bt-KKFFCAIL, and different amounts of DATFP-FPP (▾), DATFP-GPP (bullet), DATFP-DMAPP (), and DATFP-FMP ().



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 K value was only slightly higher than the estimated K of 20 nM for FPP, which was determined in the same experiment. Similarly, hPGGT-I was competitively inhibited by DATFP-FPP with a K value of 18 nM, whereas the estimated Kvalue of 16 nM was observed for its isoprenoid substrate, GGPP (Fig.2B).


Figure 2: Double reciprocal plots of the activity of hPFT and hPGGT-I in the presence of inhibitor DATFP-FPP. hPFT and hPGGT-I were assayed as described under ``Experimental Procedures'' using (A) 2 ng/ml recombinant hPFT, 10 µM Bt-KTKCVIS, variable [^3H]FPP, and 0 nM (), 66.7 nM (bullet), and 200 nM () DATFP-FPP, and (B) 1.7 ng/ml of recombinant hPGGT-I, 2 µM KKFFCAIL, variable [^3H]GGPP, and 0 nM (), 22.2 nM (bullet), and 66.7 nM () DATFP-FPP.



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 methods. No detectable product appearance (or loss of KTKCVIS) was observed by HPLC analysis (see ``Experimental Procedures'' for details) when DATFP-FPP, DATFP-GPP, or DATFP-DMAPP was used as an alternative prenyl donor. At least 5 nmol of a prenylated peptide could have been detected by this method of analysis. As a positive control, FPP and 25 nmol of unbiotinylated KTKCVIS were incubated with hPFT over a 6-h period. A time dependent loss of KTKCVIS (elution time = 17 min) and appearance of a product peak (elution time = 27 min) were observed. At the end of the incubation, the starting peptide was completely consumed. The nature of the HPLC purified product was established by MALDI-MS analysis. A parent molecular ion [M + H] of 983 mass units, which corresponds to the expected mass for a farnesylated peptide, was obtained. MALDI-MS analysis of a whole incubation mixture without subsequent HPLC purification also showed the appearance of farnesylated peptide. These experiments show that DATFP-FPP cannot serve as a substrate in the reaction catalyzed by hPFT, despite a low K value of hPFT for DATFP-FPP. One concludes then that DATFP-FPP behaved as a dead end inhibitor in the kinetic experiments shown on Fig.2A. An alternative and more sensitive method of analysis was used to test [^3H]DATFP-GPP as a potential substrate for hPFT. Incubation of hPFT with [^3H]DATFP-GPP and Bt-KTKCVIS showed no detectable radiolabeled product using the standard enzymatic assay. The conclusions from this experiment support the findings from HPLC analysis, but are tempered by the fact that DATFP-GPP was not as good an inhibitor of hPFT as DATFP-FPP.

Covalent Labeling of the beta-Subunits of hPFT and hPGGT-I with [P]DATFP-FPP

Photolysis of [P]DATFP-FPP in the presence of recombinant human protein prenyltransferases led to specific radiolabeling of the beta-subunits of both enzymes (Fig.3). In the absence of irradiation, no radioactive bands were observed (data not shown). The photolabeling was not affected by the absence of a peptide substrate (Fig.3, lanes 2`), which is consistent with the random order of substrate binding previously postulated for hPFT (37) and bovine brain PGGT-I(24) .


Figure 3: Photolabeling of human PFT and PGGT-I with [P]DATFP-FPP. hPFT (4 µg) or hPGGT-I (7 µg) were irradiated with 33.3 µM [P]DATFP-FPP (80 mCi/mmol) in 42 mM potassium phosphate buffer, pH 7.0, 3 mM MgCl(2), 33 µM ZnCl(2) for 10 min at 4 °C with or without Bt-peptide, FPP, or GGPP. Proteins were separated by SDS-polyacrylamide gel electrophoresis in 10% Tris-Tricine gels and silver stained (lanes 1-4). Labeled protein was detected by autoradiography (12 h; lanes 1`-4`). Gels with hPFT are in the leftpanels and those with hPGGT-I are in the right panels. Irradiation conditions: lanes 1 and 1`, with 33 µM Bt-KTKCVIS and Bt-KKFFCAIL, respectively; lanes 2 and 2`, without the peptides; lanes 3 and 3`, with 67 µM FPP; lanes 4 and 4`, with 67 µM GGPP.



Selective Inhibition of Photolabeling of hPFT and hPGGT-I by Prenyl Diphosphate Substrates

Preincubation of hPFT and hPGGT-I with the appropriate prenyl diphosphate substrate resulted in almost complete inhibition of photolabeling (Fig.3). The protective effect of the natural substrate (FPP in the case of hPFT, lane 3`, or GGPP for hPGGT, lane 4`) was substantially stronger than that of the alternative prenyl diphosphate. The inhibitory results described in Fig.3were obtained with relatively high concentrations of FPP and GGPP. Therefore, a more quantitative comparison of the relative effectiveness of FPP and GGPP as inhibitors of the cross-linking was evaluated at about 10 times lower concentrations of prenyl diphosphates and radiolabeled probe (Fig.4). Photolabeling of the beta-subunits of hPFT and hPGGT was strongly inhibited with 1.67 µM FPP and GGPP, respectively, while 6.7 µM GGPP and FPP in the corresponding cases had markedly less effect. The natural prenyl diphosphate substrates were clearly much better inhibitors for their respective protein prenyltransferases. This suggests that the photoprobe binds specifically to the prenyl diphosphate substrate binding domain of each enzyme.


Figure 4: Comparison of the inhibitory effect of C- and C-allylic diphosphates on the photolabeling of the hPFT and hPGGT-I by [P]DATFP-FPP. hPFT (top) and hPGGT-I (bottom), 2 µg each, were irradiated for 15 min at 4 °C with 3.3 µM [P]DATFP-FPP (1 Ci/mmol) in a 42 mM potassium phosphate buffer containing 3 mM MgCl(2), 33 µM ZnCl(2), and different concentrations of FPP and GGPP. The concentration of FPP or GGPP is shown below the graph. The alpha- (filled bars) and beta-subunits (open bars) were separated on a 10% Tris-Tricine polyacrylamide gel and counted for radioactivity.



Weak but noticeable nonspecific radiolabeling of the alpha-subunits of the enzymes was also observed (see Fig.3and Fig. 4), although the level of nonspecific labeling varied from preparation to preparation of the photoprobe. The level of alpha-subunit labeling was not affected by the presence of isoprenoid substrates in photolysis incubations and it appeared to be similar to that observed for the beta-subunits under conditions where the concentrations of inhibiting isoprenoid substrate were very high.

The level of photolabeling of the beta-subunits of both hPFT and hPGGT-I increased with increasing concentrations of [P]DATFP-FPP and was saturable (data not shown). The apparent relative affinities of the prenyltransferases for DATFP-FPP versus their natural prenyl diphosphate substrates were also estimated indirectly by determining the dependence of inhibition of the radiolabeling of the beta-subunits on the concentrations of FPP and GGPP. Prenyl diphosphate concentrations which gave 50% inhibition of beta-subunit photolabeling at 2.2 µM [P]DATFP-FPP were 0.4 µM FPP and 1.1 µM GGPP, respectively, for hPFT and hPGGT-I (data not shown). Therefore, the photoprobe binds well to both enzymes but with an affinity which is poorer compared to FPP (for hPFT) and comparable to GGPP (for hPGGT-I).


DISCUSSION

The application of photoaffinity labeling to the study of prenyltransferases offers the possibility of exploring the active sites of these enzymes. This points to the need to develop photoreactive isoprenoid analogues which react specifically with different prenyl diphosphate binding domains. Brems and Rilling (38) showed the potential of using photoreactive isoprenoid analogues as probes of prenyltransferases by demonstrating that [^3H]o-azidophenylethyl diphosphate labeled the isopentenyl diphosphate binding site of avian liver FPP synthase. Our approach has been to synthesize analogues of the allylic prenyl diphosphate substrate where the -isoprene residue was replaced by the photoreactive DATFP group. Previous work described the synthesis of diazotrifluoropropionyloxy derivatives of dimethylallyl and geranyl diphosphate (I, n = 0, 1) as analogues of GPP and FPP, respectively(21) . The planar configuration of the diazoacyl group was expected to mimic an isoprene unit having a rigid arrangement of substituents around the double bond. The effectiveness of DATFP-GPP as an inhibitory analogue of FPP has been described for several prenyltransferases(21, 22, 23, 39) . Recently, [^3H]DATFP-GPP and [^3H]DATFP-FPP have been used to photolabel the beta-subunits of recombinant hPFT (5) and purified bovine brain PGGT-I (24) , respectively.

We have now shown that both hPFT and hPGGT-I were strongly inhibited by DATFP-FPP, whereas the shorter chain analogues DATFP-GPP and DATFP-DMAPP were much poorer inhibitors. Furthermore, the monophosphate ester of the geranylgeranyl analogue, DATFP-FMP, was also a poor inhibitor. One would conclude then that the diphosphate moiety and a lipid moiety, at least as long as the DATFP-farnesyl group, are necessary for exhibiting strong inhibitory properties. Surprisingly, DATFP-FPP, the putative GGPP analogue, was a better inhibitor of hPFT than the FPP analogue DATFP-GPP. This might suggest that hPFT recognizes the farnesyl moiety of DATFP-FPP and binds it more tightly than the DATFP-geranyl moiety of DATFP-GPP.

The apparent binding affinities of DATFP-FPP for hPFT and hPGGT-I were estimated by two methods: 1) kinetic analysis of inhibition of prenyltransferase activity by the photoprobe and 2) analysis of the concentration-dependent inhibition of enzyme photolabeling by the natural substrates FPP and GGPP. DATFP-FPP was a competitive inhibitor of both hPFT and hPGGT-I. The K (100 nM) of hPFT for DATFP-FPP, determined kinetically, was 5 times higher than the K for FPP, whereas, the K (18 nM) of hPGGT-I for DATFP-FPP was about the same as the Kfor GGPP (Table1). The K values observed here are comparable to those previously reported for these enzymes(5, 34) . Analyses of probe affinity for the enzymes by the indirect method, where inhibition of photolabeling of hPFT and hPGGT-I by FPP and GGPP was assessed, also showed that relative differences in affinity between DATFP-FPP and the natural substrates were similar to the ratios of K/K for these compounds. Therefore, the affinities of DATFP-FPP for hPFT and hPGGT-I were somewhat less than either of the natural substrates. Nevertheless, DATFP-FPP binds competitively and tightly to both enzymes.

The mode of involvement of protein subunits or their respective amino acids residues in the active sites of prenyltransferases in general has not been established, although the first crystal structure for a prenyltransferase, FPP synthase, has now been reported(40) . Radiolabeled photoanalogues of prenyltransferase substrates have the potential to identify key features of the protein structure of prenyltransferases. The FPP analogue, [^3H]DATFP-GPP, has already been shown to photolabel two other prenyltransferases(22, 41) in addition to the beta-subunits of hPFT (5) and hPGGT-I(24) . The synthesis of the GGPP analogues, [^3H]- and [P]DATFP-FPP, has now made it possible to also photolabel purified bovine brain (24) and recombinant human PGGT-I. Heavy labeling of the beta-subunits of these enzymes, in contrast to the weaker nonspecific labeling of the alpha-subunits, points to the specificity of the cross-linking procedure. Effective inhibition of labeling of the respective prenyltransferases by their prenyl diphosphate substrates and poor inhibition by the prenyl diphosphate substrate of the other protein prenyltransferase clearly shows that the photoprobe is directed toward the active site or ``substrate binding site'' and not some other hydrophobic environment on the beta-subunit. Therefore, demonstrating that DATFP-FPP is a competitive inhibitor of the natural allylic diphosphate substrates for the protein prenyltransferases and showing that inhibition of labeling of the beta-subunits of both hPFT and hPGGT-I is specific for the prenyl diphosphate substrate leads to the conclusion that the beta-subunits of these protein prenyltransferases are involved in the specific recognition and binding of the prenyl diphosphate substrate. The mode of association of the prenyl diphosphate with subunits of rab PGGT still remains to be established.

The labeling of the beta-subunit with the isoprenoid related probes is consistent with previous photo-cross-linking results which showed that both peptide and protein substrates can interact with the beta-subunit of PFT(42, 43) . These probes now offer the opportunity to identify specific amino acid residues in the isoprenoid and peptide binding domains of these enzymes. The P-labeled photoprobes offer some advantages over the ^3H-labeled probes for peptide localization and analysis because they are more readily detectable. In addition, the P-labeled probes can be easily prepared because phosphorylation is the last step in the synthesis. Identification of cross-linked amino acid residues would provide potential targets for a variety of approaches to precisely define critical residues which are involved in catalysis and determining the specificity of isoprenoid substrate binding. Such approaches might include site-directed mutagenesis and the design of other reactive prenyl diphosphate analogues. Since both hPFT and hPGGT-I show a tolerance for alterations in the structure of the -isoprene unit when binding prenyl diphosphate analogues, new isoprenoid based analogues might be designed with more effective and site-specific functional groups attached at the -terminus. One approach could be to incorporate substituents which are chemically reactive with specific amino acid residues. One can also envision the use of such photoprobes to map the binding sites of other prenyltransferases and enzymes that metabolize prenyl diphosphates. The synthesis of appropriate isoprenoid analogues could bring new perspective to studies on proteins involved in prenylated protein biosynthesis and function, such as COOH-terminal proteases, methyl transferases, and prenylated protein binding proteins.


FOOTNOTES

*
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 by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

^1
The abbreviations used are: PFT, protein farnesyltransferase; Bt, biotinylated; DATFP, diazotrifluoropropionyloxy; GPP, geranyl diphosphate; DMAPP, dimethylallyl diphosphate; FMP, farnesyl monophosphate; FPP, farnesyl 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 chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

600 MHz nmr DQFCOSY and NOESY experiments were carried out by John West at the Center for Structural Biology, University of Florida. MADLI-MS analysis was done by Huang P. Nguyen, Protein Chemistry Core of ICBR, University of Florida.


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