Mapping of the Catalytic Groove Preferences of Factor Xa Reveals an Inadequate Selectivity for Its Macromolecule Substrates
2002; Elsevier BV; Volume: 277; Issue: 23 Linguagem: Inglês
10.1074/jbc.m201139200
ISSN1083-351X
AutoresElsa P. Bianchini, Virginie B. Louvain, Pierre-Emmanuel Marque, María A. Juliano, Luiz Juliano, Bernard Le Bonniec,
Tópico(s)Hemophilia Treatment and Research
ResumoFactor Xa (FXa) hydrolyzes two peptide bonds in prothrombin having (Glu/Asp)-Gly-Arg-(Thr/Ile) for P3-P2-P1-P1′ residues, but the exact preferences of its catalytic groove remain largely unknown. To investigate the specificity of FXa, we synthesized full sets of fluorescence-quenched substrates carrying all natural amino acids (except Cys) in P3, P2, P1′, P2′, and P3′ and determined the kcat /Km values of cleavage. Contrary to expectation, glycine was not the “best” P2 residue; peptide with phenylalanine was cleaved slightly faster. In fact, FXa had surprisingly limited preferences, barely more pronounced than trypsin; in P2, the ratio of the kcat /Km values for the most favorable side chain over the least was 289 (12 with trypsin), but in P1′, this ratio was only 30 (versus 80 with trypsin). This unexpected selectivity undoubtedly distinguished FXa from thrombin, which exhibited ratios higher than 19,000 in P2 and P1′. Thus, with respect to the catalytic groove, FXa resembles a low efficiency trypsin rather than the highly selective thrombin. The rates of cleavage of the peptidyl substrates were virtually identical whether or not FXa was in complex with factor Va, suggesting that the cofactor did not exert a direct allosteric control on the catalytic groove. We conclude that the remarkable efficacy of FXa within prothrombinase originates from exosite interaction(s) with factor Va and/or prothrombin rather than from the selectivity of its catalytic groove. Factor Xa (FXa) hydrolyzes two peptide bonds in prothrombin having (Glu/Asp)-Gly-Arg-(Thr/Ile) for P3-P2-P1-P1′ residues, but the exact preferences of its catalytic groove remain largely unknown. To investigate the specificity of FXa, we synthesized full sets of fluorescence-quenched substrates carrying all natural amino acids (except Cys) in P3, P2, P1′, P2′, and P3′ and determined the kcat /Km values of cleavage. Contrary to expectation, glycine was not the “best” P2 residue; peptide with phenylalanine was cleaved slightly faster. In fact, FXa had surprisingly limited preferences, barely more pronounced than trypsin; in P2, the ratio of the kcat /Km values for the most favorable side chain over the least was 289 (12 with trypsin), but in P1′, this ratio was only 30 (versus 80 with trypsin). This unexpected selectivity undoubtedly distinguished FXa from thrombin, which exhibited ratios higher than 19,000 in P2 and P1′. Thus, with respect to the catalytic groove, FXa resembles a low efficiency trypsin rather than the highly selective thrombin. The rates of cleavage of the peptidyl substrates were virtually identical whether or not FXa was in complex with factor Va, suggesting that the cofactor did not exert a direct allosteric control on the catalytic groove. We conclude that the remarkable efficacy of FXa within prothrombinase originates from exosite interaction(s) with factor Va and/or prothrombin rather than from the selectivity of its catalytic groove. At the confluence of the formerly named intrinsic and extrinsic pathways, factor Xa (FXa) 1The abbreviations used are: FXafactor XaRVV-XFX-activating enzyme from Russell's viper venomTFPItissue factor pathway inhibitor (Kunitz type 1)pNApara-nitroanilineS-2222benzoyl-Ile-Glu(γ-OR)-Gly-Arg-pNAABzortho-amino-benzoylEDDnpN-(2,4-dinitrophenyl) ethylenediamine is the midway protease of the blood clotting waterfall (1Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1626) Google Scholar). FXa belongs to clan SA of the S1 family of serine peptidases along with thrombin and trypsin (2Padmanabhan K. Padmanabhan K.P. Tulinsky A. Park C.H. Bode W. Huber R. Blankenship D.T. Cardin A.D. Kisiel W. J. Mol. Biol. 1993; 232: 947-966Crossref PubMed Scopus (401) Google Scholar, 3Brandstetter H. Kuhne A. Bode W. Huber R. Von Der Saal W. Wirthensohn K. Engh R.A. J. Biol. Chem. 1996; 271: 29988-29992Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 4Bode W. Brandstetter H. Mather T. Stubbs M.T. Thromb. Haemostasis. 1997; 78: 501-511Crossref PubMed Scopus (99) Google Scholar, 5Rawlings N.D Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes: Family S1 of Trypsin. Academic Press, London, UK1998: 3-17Google Scholar). Without cofactors, activation of prothrombin by FXa is slow; it becomes efficient only when FXa complexes factor Va to form prothrombinase (6Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar). Rapid inhibition of FXa by antithrombin also requires heparin as cofactor (7Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology. R. G. Landes, Austin, TX1996Google Scholar). However, tissue factor pathway inhibitor (TFPI) does not require any cofactor to rapidly neutralize FXa (8Baugh R.J. Broze G.J., Jr. Krishnaswamy S. J. Biol. Chem. 1998; 273: 4378-4386Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). FXa catalyzes a number of other reactions: activation of factor VII in a positive feedback within the tissue factor pathway (9Bajaj S.P. Rapaport S.I. Brown S.F. J. Biol. Chem. 1981; 256: 253-259Abstract Full Text PDF PubMed Google Scholar), activation of factors V (10Foster W.B. Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 13970-13977Abstract Full Text PDF PubMed Google Scholar) and VIII (11Parker E.T. Pohl J. Blackburn M.N. Lollar P. Biochemistry. 1997; 36: 9365-9373Crossref PubMed Scopus (25) Google Scholar), cleavage of protease-activated receptor 2 (12Camerer E. Huang W. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5255-5260Crossref PubMed Scopus (610) Google Scholar), and neutralization of protein S, albeit only in the presence of phospholipid and calcium (13Long G.L., Lu, D. Xie R.L. Kalafatis M. J. Biol. Chem. 1998; 273: 11521-11526Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Thrombin (14Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar) requires a cofactor for activation of protein C and factor XI, as well as for its inhibition by antithrombin and heparin cofactor II. In contrast to FXa, however, thrombin alone rapidly catalyzes a number of critical reactions in the cascade: cleavage of fibrinogen, activation of factors V and VIII, and activation of protease-activated receptor 1 (6Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar, 10Foster W.B. Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 13970-13977Abstract Full Text PDF PubMed Google Scholar, 15Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2140) Google Scholar). Trypsin, the archetypal endopeptidase of the digestive tract, does not require cofactors to rapidly hydrolyze (in appropriate conditions) most peptide bonds that follow an arginine or a lysine. The notable specificity of the blood coagulation peptidases result from at least four molecular mechanisms: (i) constraints built up by subsites 3 to 3′ (S3 to S3′) 2The residues of the substrates are numbered from P6 to P6′, where P6 and P6′ refer to six residues remote from the cleavage site on the N- and C-terminal side, respectively. The corresponding subsites on the enzyme are numbered from S6 to S6′. of the catalytic groove, which accommodate the P3–P3′ residues of the substrate or inhibitor; (ii) steric restrictions caused by surface loops surrounding the catalytic groove; (iii) exosites remote from the catalytic groove, which may anchor complementary motifs of the overall substrate protein; and (iv) cofactors that may overturn the specificity of the protease (4Bode W. Brandstetter H. Mather T. Stubbs M.T. Thromb. Haemostasis. 1997; 78: 501-511Crossref PubMed Scopus (99) Google Scholar, 16Le Bonniec B.F. Myles T. Johnson T. Knight C.G. Tapparelli C. Stone S.R. Biochemistry. 1996; 35: 7114-7122Crossref PubMed Scopus (89) Google Scholar, 17Stubbs M.T. Oschkinat H. Mayr I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (198) Google Scholar, 18Mathews I.I. Padmanabhan K.P. Ganesh V. Tulinsky A. Ishii M. Chen J. Turck C.W. Coughlin S.R. Fenton J.W., II Biochemistry. 1994; 33: 3266-3279Crossref PubMed Scopus (166) Google Scholar, 19Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (451) Google Scholar, 20Banner D.W. D'Arcy A. Chène C. Winkler F.K. Guha A. Konigsberg W.H. Nemerson Y. Kirchhofer D. Nature. 1996; 380: 41-46Crossref PubMed Scopus (686) Google Scholar, 21Fuentes-Prior P. Iwanaga Y. Huber R. Pagila R. Rumennik G. Seto M. Morser J. Light D.R. Bode W. Nature. 2000; 404: 518-525Crossref PubMed Scopus (279) Google Scholar). factor Xa FX-activating enzyme from Russell's viper venom tissue factor pathway inhibitor (Kunitz type 1) para-nitroaniline benzoyl-Ile-Glu(γ-OR)-Gly-Arg-pNA ortho-amino-benzoyl N-(2,4-dinitrophenyl) ethylenediamine A comparison of the P3–P3′ sequences of the known substrates and inhibitors of FXa suggests that glycine in P2 and serine in P1′ could favor catalysis. In this paper, we report a comprehensive study of FXa subsites preferences using fluorescence-quenched substrates. We also compared FXa preferences with those of thrombin and trypsin. Some of the subsite preferences of FXa were unexpected, but the main surprise came from the overall limited selectivity of its catalytic groove. Addition of factor Va, phospholipid, and calcium had no detectable influence on FXa preferences or on its catalytic efficiency. Thus, we conclude that the remarkable efficacy of FXa within prothrombinase must rely on exosite(s) interaction(s) rather than on a purely allosteric mechanism involving its catalytic groove. Prothrombin was purified from human plasma and converted to thrombin as described previously (22Le Bonniec B.F. MacGillivray R.T.A. Esmon C.T. J. Biol. Chem. 1991; 266: 13796-13803Abstract Full Text PDF PubMed Google Scholar). Bovine factor V/Va was from Kordia (Leiden, The Netherlands), and phospholipids vesicles were prepared by sonication of a 1 mg/ml mixture of phosphatidylcholine (80% w/w) with phosphatidylserine (20% w/w) (Sigma) as described previously (23Le Bonniec B.F. Guinto E.R. Esmon C.T. J. Biol. Chem. 1992; 267: 6970-6976Abstract Full Text PDF PubMed Google Scholar). Human FX cDNA (24Messier T.L. Pittman D.D. Long G.L. Kaufman R.J. Church W.R. Gene (Amst.). 1991; 99: 291-294Crossref PubMed Scopus (40) Google Scholar) was amplified by polymerase chain reaction using primers ACG-CGG-ATC-CGC-GAT-GGG-GCG-CCC-ACT-GCA and TCC-CCC-GGG-GGA-TCA-GTT-CAG-GTC-TTC-CTC-GCT-GAT-CAG-CTT-CTG-CTC-CTT-TAA-TGG-AGA-GGA-CGT-TA, which introduced a BamHI restriction site at the 5′ end and an XmaI site at the 3′ end, respectively. The 3′ end primer also inserted in-frame with the C terminus of the FX cDNA a sequence coding for the epitope EEQKLISEEDLLGGY recognized by monoclonal antibody 9E10. The insert between the BamHI and XmaI sites of plasmid pNUT-hGH (22Le Bonniec B.F. MacGillivray R.T.A. Esmon C.T. J. Biol. Chem. 1991; 266: 13796-13803Abstract Full Text PDF PubMed Google Scholar) was substituted for the product of amplification, and the full-length cDNA sequence was verified by dideoxy chain termination sequencing. Transfection and selection of BHK-21 cells were achieved as described previously (22Le Bonniec B.F. MacGillivray R.T.A. Esmon C.T. J. Biol. Chem. 1991; 266: 13796-13803Abstract Full Text PDF PubMed Google Scholar,25Le Bonniec B.F. Guinto E.R. MacGillivray R.T.A. Stone S.R. Esmon C.T. J. Biol. Chem. 1993; 268: 19055-19061Abstract Full Text PDF PubMed Google Scholar). Production and purification of recombinant FX were also performed as described previously, except that c-Myc monoclonal antibody-agarose (CLONTECH, Saint Quentin, France) was used for the purification. Recombinant FX was eluted by 100 mmglycine-HCl, pH 2.7. The pH was immediately neutralized by adding 30 μl/ml of 2 m Tris, and FX was further purified (after dilution 1:4 in 50 mm Tris-HCl, pH 7.5, containing 5 mm EDTA) by anion exchange chromatography onto Q-Sepharose fast flow (Amersham Biosciences) developed with a linear gradient from 0.15 to 0.5 m NaCl in 50 mm Tris-HCl, pH 7.5. Recombinant FX was converted to FXa essentially according to Jesty and Nemerson (26Jesty J. Nemerson Y. Methods Enzymol. 1976; 45: 95-107Crossref PubMed Scopus (75) Google Scholar) by incubation with the activator purified from the Russell's viper venom (RVV-X) purchased from Kordia. Briefly, RVV-X (5 mg/ml) in 200 mm NaHCO3, pH 8.3, containing 0.5m NaCl was coupled to a 5-ml HiTrap N-hydroxysuccinimide-activated column (AmershamBiosciences) following the manufacturer's instructions. The column was sealed after loading with 4 mg of FX in 4 ml of kinetic buffer (50 mm Tris-HCl, pH 7.5, containing 0.15 m NaCl, 5 mm CaCl2, and 0.2% (w/v) poly(ethylene glycol); Mr = 6000), and the incubation was prolonged for 16 h at room temperature. The eluate, diluted 1:3 in 50 mm Tris-HCl, pH 7.5, containing 5 mmCaCl2, was loaded onto a 1-ml HiTrap heparin-Sepharose column (Amersham Biosciences) and eluted with 50 mmTris-HCl, pH 7.5, containing 0.5 m NaCl and 5 mm CaCl2. Recombinant FXa appeared pure by SDS-polyacrylamide gel analysis and was indistinguishable from human FXa commercialized by Kordia with respect to its Km and kcat values for the hydrolysis of benzoyl-Ile-Glu(γ-OR)-Gly-Arg-pNA (S-2222, Biogenic, Maurin, France), Z-d-Arg-Gly-Arg-pNA (Biogenic), as well as the rate of prothrombin activation within prothrombinase. Recombinant and commercial FXa were nevertheless systematically compared in all experiments reported in this study and found virtually identical despite the C-terminal 9E10 epitope. The active site concentration of a stock trypsin solution (bovine, tosylphenylalanyl chloromethyl ketone-treated; Worthington, Lorne Laboratories, Twyford, UK) was determined by titration with p- nitrophenyl-p′-guanidinobenzoate. This titrated trypsin was used to determine the precise concentration of aliquots in 1 mm HCl of d-Phe-Phe-Arg-CH2Cl andd-Phe-Pro-Arg-CH2Cl (Calbiochem, Meudon, France). Briefly, 200 nm trypsin in kinetic buffer was incubated for 3 h at room temperature with various amount of each chloromethyl ketone (0.03–3 μm). The reaction mixture was diluted 1:10 in kinetic buffer containing 100 μmS-2222, and the remaining enzyme concentration was estimated from the rate of A405 increase. The initial concentrations of chloromethyl ketone aliquots were deduced from the intercept to the x axis of a linear plot of the remaining activity versus the amount of inhibitor added. The active site concentrations of FXa (recombinant or human) and of thrombin were determined in the same buffer system and experimental conditions using the calibrated aliquots of chloromethyl ketone. For FXa titration, 1 μm enzyme according to the A280(extinction coefficient of 1.25 ml mg−1 cm−1) was incubated with 0.1–5 μm d-Phe-Phe-Arg-CH2Cl, and the remaining free enzyme concentration was measured with 100 μm S-2222 as substrate. For thrombin titration, 20 nm enzyme according to the A280 (extinction coefficient of 2.0 ml mg−1 cm−1) was incubated with 2–200 nm d-Phe-Pro-Arg-CH2Cl, and the remaining free enzyme concentration was measured with 100 μm H-d-Phe-pipecolyl-Arg-pNA (Biogenic). Just prior to use, FXa and trypsin dilutions were systematically controlled by measuring the rate of S-2222 hydrolysis, and thrombin dilutions were controlled by measuring the rate of H-d-Phe-pip-Arg-pNA hydrolysi s. Fluorescence-quenched substrates were prepared by the solid phase method via a Fmoc (N-(9-fluorenyl)methoxycarbonyl)/t-butyl strategy, purified by reverse phase chromatography on a C18 column, and their purity was checked by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Tof-Spec-E, Micromass) as described previously (27Hirata I.Y. Cesari M.H.S. Nakaie C.R. Boschcov P. Ito A.S. Juliano M.A. Juliano L. Lett. Peptide Sci. 1994; 1: 299-301Crossref Scopus (200) Google Scholar). pNA substrates were prepared by the solution method following the procedure described in Alves et al. (28Alves L.C. Almeida P.C. Franzoni L. Juliano L. Juliano M.A. Peptide Res. 1996; 9: 92-96PubMed Google Scholar). Lyophilized substrates were resuspended in a minimum volume N, N-dimethylformamide, such that concentration in the stock solutions was about 5 mm, to ensure that during all kinetic experiments, the final amount of N, N-dimethylformamide never exceeded 0.5% (v/v). The concentration of the fluorescence-quenched substrates was estimated from their A360, assuming an absorption coefficient of 104 m −1cm−1. Determination of the kcat /Km values of hydrolysis was performed essentially as described previously (16Le Bonniec B.F. Myles T. Johnson T. Knight C.G. Tapparelli C. Stone S.R. Biochemistry. 1996; 35: 7114-7122Crossref PubMed Scopus (89) Google Scholar, 29Marque P.E. Spuntarelli R. Juliano L. Aiach M. Le Bonniec B.F. J. Biol. Chem. 2000; 275: 809-816Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Assuming that the reaction obeys a simple Michaelis-Menten mechanism with the encounter of enzyme and substrate limiting, when the reaction is performed at a substrate concentration below Km, the progress curve kinetic allows estimation of the kcat/Km of the reaction. Briefly, hydrolysis at 37 °C was monitored by measuring the fluorescence at λem = 414 nm (slit, 4 nm) and λex = 325 nm (slit, 10 nm) in a LS50B spectrofluorimeter (PerkinElmer Life Sciences) equipped with a thermostatted microplate reader. A microplate containing 180 μl of substrate (1–8 μm) in kinetic buffer containing 0.2% (v/v) Tween 20 was left in the thermostatted compartment until the temperature equilibrium was attained (10–15 min), and the reaction was started by adding 20 μl of enzyme (0.1–200 nm). Care was taken to keep the ionic strength constant, in particular the NaCl concentration (30Lottenberg R. Hall J.A. Pautler E Zupan A. Christensen U. Jackson C.M. Biochim. Biophys. Acta. 1986; 874: 326-336Crossref PubMed Scopus (30) Google Scholar, 31Dang Q.D. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10653-10656Crossref PubMed Scopus (145) Google Scholar, 32Underwood M.C. Zhong D. Mathur A. Heyduk T. Bajaj S.P. J. Biol. Chem. 2000; 275: 36876-36884Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The increase in fluorescence with time was monitored for up to 4 h (until at least 75% completion of the reaction). The pseudo-first order rate constant (k), the initial fluorescence (Io), and the maximum fluorescence intensity (Imax) were estimated by nonlinear curve fitting analysis of the fluorescence intensity (INT) dependence on time (t) using the following equation.INT=Io+Imax(1−exp−E k t)Equation 1 where E is the enzyme concentration. The values of Io and Imax estimated through the nonlinear curve fitting analysis were always consistent with the fluorescence intensities measured before and after 1 h of incubation of the substrate with 0.1 μm trypsin (corresponding to the intact and fully cleaved peptide, respectively). The apparent rate constant will be pseudo-first order and will equal kcat /Km, provided that the initial substrate concentration is much less than its Km value for the enzyme. To assess whether this condition was met, progress curve kinetics were systematically performed at two initial concentrations of fluorescence-quenched substrates. the results indicated that with all of the substrates assayed k was, within the experimental error, identical at the two substrate concentrations. Thus, in this concentration range, the rate of hydrolysis was not dependent upon the amount of substrate, suggesting that k could be equated to the kcat /Km value. When substrates carried several potential cleavage sites, the preferred bond of hydrolysis was determined after purification of the products by reverse phase chromatography. Kinetics were performed in conditions identical to those used for the fluorescence studies except that the reactions were quenched at timed intervals by adding H3PO4 (1% w/v). The aliquots were loaded on a 250 × 4.6 mm Vydac 218TP54 column (Interchim, Asnières, France) developed with a 10–80% linear gradient acetonitrile in 0.5% (w/v) H3PO4. The peaks were identified by monitoring the A254 and A360 and were quantified by integration analysis; their identity was confirmed by N-terminal sequencing. Estimation of the rate constant for formation of each product was obtained by data analysis using Equation 1, in which the initial, sampling time, and final A360 (or A254), was substituted for Io, INT, and Imax, respectively. Kinetics of pNA substrate hydrolysis by FXa (100 nm) were performed in a microplate at 25 °C and monitored by measuring the A405 in a model MR 5000 reader (Dynex, Guyancourt, France). The concentrations of the pNA substrates (10 and 20 μm) were adjusted according to their A316, assuming an absorption coefficient of 12,700 m −1 cm−1. The data were analyzed using Equation 1 in which the A405 values (initial, at time t, and final) were substituted for Io, INT, and Imax, respectively. With all of the pNA substrates examined, the apparent rate constants of cleavage obtained at 10 and 20 μm substrate were indistinguishable. Thus, the Km for FXa was likely higher than 200 μm, allowing the pseudo-first order rate constant to be equated to kcat /Km. The values reported represent the weighted means of at least three determinations. To investigate the catalytic groove preferences of FXa, we synthesized five series of fluorescence-quenched substrates, having a common 10-amino acid-long framework (ABz-VQFRSLGDQ-EDDnp). The peptides carried a strongly fluorescent ABz group at the N-terminal end, but a C-terminal EDDnp quenched this fluorescence by resonance energy transfer. The kcat /Km value for the cleavage of each peptide by FXa was estimated by analysis of the increase of the fluorescence intensity upon hydrolysis. In each series of peptides, either the P3, P2, P1′, P2′, or P3′ amino acid was varied, such that complete sets of peptides were prepared covering all natural amino acids (except cysteine). Proline in P1′ was also avoided because it is known to prohibit trypsin cleavage. Study of the P2 preferences of FXa revealed that, contrary to expectation, glycine was not the most favorable amino acid; the peptide having a phenylalanine residue in P2 was cleaved slightly faster than the one with a glycine (Table I). Overall, substrates with aromatic side chains in P2 were cleaved efficiently, whereas the least favorable amino acid was aspartate. The FXa cleavage sites in prothrombin and factor VII have glycine in P2. In fact, a number of studies have established that FXa prefers Gly over Phe in P2 position with chromogenic or fluorogenic substrates (30Lottenberg R. Hall J.A. Pautler E Zupan A. Christensen U. Jackson C.M. Biochim. Biophys. Acta. 1986; 874: 326-336Crossref PubMed Scopus (30) Google Scholar,33Lottenberg R. Christensen U. ackson C.M. Coleman P.L. Methods Enzymol. 1981; 80: 341-361Crossref PubMed Scopus (338) Google Scholar, 34McRae B.J. Kurachi K. Heimark R.L. Fujikawa K. Davie E.W. Powers J.C. 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Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes: Family S1 of Trypsin. Academic Press, London, UK1998: 3-17Google Scholar). normally blocks S2 of FXa (2Padmanabhan K. Padmanabhan K.P. Tulinsky A. Park C.H. Bode W. Huber R. Blankenship D.T. Cardin A.D. Kisiel W. J. Mol. Biol. 1993; 232: 947-966Crossref PubMed Scopus (401) Google Scholar, 3Brandstetter H. Kuhne A. Bode W. Huber R. Von Der Saal W. Wirthensohn K. Engh R.A. J. Biol. Chem. 1996; 271: 29988-29992Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Thus, it was quite surprising that bulky side chains (phenylalanine and tryptophan) could be as efficient as glycine in our study. The discrepancy could originate from the influence of the P3/P4 apolard-amino acid present in the substrates used in most previous studies on FXa specificity. Binding of a P3/P4 d-amino acid is highly favorable and clearly improves the catalytic efficiency of FXa (30Lottenberg R. Hall J.A. Pautler E Zupan A. Christensen U. Jackson C.M. Biochim. Biophys. Acta. 1986; 874: 326-336Crossref PubMed Scopus (30) Google Scholar). It is conceivable that binding of a d-side chain in S3/S4 distorts the neighboring S2, hence prohibiting the binding of a bulky P2 side chain. On the other hand, as opposed to small P4–P1peptidyl substrates, our fluorescence-quenched substrates bind to subsites on both sides of the scissile bond. Thus, the observed specificity could also result from cooperative effects that would influence the P2 preferences of FXa. To investigate such possible cooperative effects, we synthesized a series of chromogenic substrates (acetyl-VQ XR-pNA, where X was F, T, G, or P). However, analysis of the progress curves of cleavage confirmed that, when constituted exclusively byl-amino acids, a chromogenic substrate having phenylalanine in P2 is hydrolyzed slightly faster than its counterpart with glycine (4.1 ± 0.2 versus 3.4 ± 0.2 × 104 m −1 s−1). Another possibility is that binding provides the necessary energy to move the aromatic ring system of FXa (Phe174, Tyr99, and Trp215), perhaps by a simple rotation of Tyr99toward S3/S4, as is observed in kallikrein and factor IXa (2Padmanabhan K. Padmanabhan K.P. Tulinsky A. Park C.H. Bode W. Huber R. Blankenship D.T. Cardin A.D. Kisiel W. J. Mol. Biol. 1993; 232: 947-966Crossref PubMed Scopus (401) Google Scholar, 3Brandstetter H. Kuhne A. Bode W. Huber R. Von Der Saal W. Wirthensohn K. Engh R.A. J. Biol. Chem. 1996; 271: 29988-29992Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 38Bode W. Chen Z.G. Bartels K. Kutzbach C. Schmidt-Kastner G. Bartunik H. J. Mol. Biol. 1983; 164: 237-282Crossref PubMed Scopus (223) Google Scholar, 39Hopfner K.P. Brandstetter H. Karcher A. Kopetzki E. Huber R. Engh R.A. Bode W. EMBO J. 1997; 16: 6626-6635Crossref PubMed Scopus (65) Google Scholar). Such movement would be prohibited when ad-amino acid side chain occupies S3/S4. In support of this hypothesis, FXa selects sequences with phenylalanine or tyrosine in P2 (in addition to those having glycine) when offered a library of fusion proteins displayed on phages as substrates (40Matthews D.J. Wells J.A. Science. 1993; 260: 1113-1117Crossref PubMed Scopus (317) Google Scholar) or a library of combinatorial fluorogenic substrates (41Harris J.L. Backes B.J. Leonetti F. Mahrus S. Ellman J.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7754-7759Crossref PubMed Scopus (475) Google Scholar). In addition, binding of TFPI evidently requires that Tyr99 of FXa swing away from the “normal” S2 (42Burgering M.J.M. Orbons L.P.M. van der Doelen A. Mulders J. Theunissen H.J.M. Grootenhuis P.J.M. Bode W. Huber R. Stubbs M.T. J. Mol. Biol. 1997; 269: 395-407Crossref PubMed Scopus (72) Google Scholar). Finally, in the study of Castillo et al. (43Castillo M.J. Kurachi K. Nishino N. Ohkubo I. Powers J.C. Biochemistry. 1983; 22: 1021-1029Crossref PubMed Scopus (35) Google Scholar) that also used fluorescence-quenched substrates, Gly as the “best” P2 amino acid for FXa was only measured in comparison with substrates with Val, Ser, or Thr at this position.Table IValues of kcat /Km for the cleavage by FXa of the fluorescence-quenched substratesP3kcat/KmP2kcat/KmP1′kcat/KmP2′kcat/KmP3′kcat/KmQ2.6 × 104F2.6 × 104S2.6 × 104L2.6 × 104S3.0 × 104G2.6 × 104G2.1 × 104A1.9 × 104G1.9 × 104H2.9 × 104H2.5 × 104W1.2 × 104T1.5 × 104F1.8 × 104T2.6 × 104V2.0 × 104Y7.4 × 103G9.1 × 103I1.8 × 104N2.6 × 104R1.9 × 104P5.1 × 103V7.8 × 103Y1.8 × 104G2.6 × 104W1.9 × 104A4.0 × 103N5.8 × 103A1.6 × 104D2.5 × 104A1.7 × 104L2.6 × 103H5.8 × 103V1.5 × 104K2.0 × 104F1.7 × 104S1.9 × 103Y3.7 × 103S1.4 × 104M1.8 × 104N1.6 × 104R1.7 × 103I3.3 × 103W1.2 × 104R1.8 × 104L1.6 × 104V1.7 × 103F3.2 × 103T1.2 × 104A1.8 × 104M1.4 × 104Q1.5 × 103L3.1 × 103N1.1 × 104E1.7 × 104T1.4 × 104E1.2 × 103Q2.6 × 103R8.6 × 103Q1.6 × 104S1.3 × 104H1.2 × 103R2.6 × 103H8.5 × 103P1.4 × 104K1.3 × 104T1.1 × 103M2.4 × 103E8.1 × 103Y1.0 × 104P1.3 × 104N1.1 × 103D2.4 × 103D8.1 × 103I9.0 × 103I1.2 × 104I8.3 × 102K1.9 × 103Q6.7 × 103F8.1 × 103E1.1 × 104M7.4 × 102W1.0 × 103K6.1 × 103V7.5 × 103Y6.0 × 103K3.9 × 102E8.6 × 102M4.1 × 103L7.2 × 103D4.6 × 103D9.0 × 101PNDP1.4 × 103W5.3 × 103The five series of fluorescence-quenched substrates had ABz-VQFRSLGDQ-EDDnp for a common framework. The values reported were obtained for the hydrolysis of the substrates by recombinant FXa and represent the weighted mean of at least three determinations (standard error was 18% or less). Cleavage always occurred between the arginine and the serine, even when a second arginine (or a lysine) was in the sequence. The substrate with proline in P1′ (resistant to trypsin hydrolysis) was
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