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• W2091001049 abstract "Members of the serpin (serine protease inhibitor) family share a similar backbone structure but expose a variable reactive-site loop, which binds to the catalytic groove of the target protease. Specificity originates in part from the sequence of this loop and also from secondary binding sites that contribute to the inhibitor function. To clarify the intrinsic contribution of the reactive-site loop, α1-antichymotrypsin has been utilized as a scaffold to construct chimeras carrying the loop of antithrombin III, protease nexin 1, or α1-antitrypsin. Reactive-site loops not only vary in sequence but also in length; therefore, the length of the reactive-site loop was also varied in the chimeras. The efficacy of the specificity transfer was evaluated by measuring the stoichiometry of the reaction, the ability to form an SDS-stable complex, and the association rate constant with a number of potential targets (chymotrypsin, neutrophil elastase, trypsin, thrombin, factor Xa, activated protein C, and urokinase). Overall, substitution of a reactive-site loop was not sufficient to transfer the specificity of a given serpin to α1-antichymotrypsin. Specificity of the chimera partly matched that of the loop donor and partly that of the acceptor, whereas the behavior as an inhibitor or a substrate depended upon the targeted protease. Results suggest that, aside from the contributions of the loop sequence and the framework-specific secondary binding sites, an intramolecular control may be essential for productive interaction. Members of the serpin (serine protease inhibitor) family share a similar backbone structure but expose a variable reactive-site loop, which binds to the catalytic groove of the target protease. Specificity originates in part from the sequence of this loop and also from secondary binding sites that contribute to the inhibitor function. To clarify the intrinsic contribution of the reactive-site loop, α1-antichymotrypsin has been utilized as a scaffold to construct chimeras carrying the loop of antithrombin III, protease nexin 1, or α1-antitrypsin. Reactive-site loops not only vary in sequence but also in length; therefore, the length of the reactive-site loop was also varied in the chimeras. The efficacy of the specificity transfer was evaluated by measuring the stoichiometry of the reaction, the ability to form an SDS-stable complex, and the association rate constant with a number of potential targets (chymotrypsin, neutrophil elastase, trypsin, thrombin, factor Xa, activated protein C, and urokinase). Overall, substitution of a reactive-site loop was not sufficient to transfer the specificity of a given serpin to α1-antichymotrypsin. Specificity of the chimera partly matched that of the loop donor and partly that of the acceptor, whereas the behavior as an inhibitor or a substrate depended upon the targeted protease. Results suggest that, aside from the contributions of the loop sequence and the framework-specific secondary binding sites, an intramolecular control may be essential for productive interaction. Serpins 1The abbreviations used are: serpin, serine protease inhibitor; ACT, recombinant α1-antichymotrypsin; ACT(P1=Arg), ACT where the P1 residue (Leu358) has been replaced by an arginine; ACT(P1=Arg; P2=Gly), ACT(P1=Arg; P2=Pro), and ACT(P1=Arg; P2=Ala), ACT mutant having an arginine for P1 residue and a glycine for P2 residue (proline and alanine for P2residue, respectively); ACT(des-TIVR), ACT where residues Thr366-Arg369 (positions P8′ to P11′ of the reactive-site loop) have been deleted; ACT/ATIII, chimera in which the ACT framework carries the reactive-site loop of antithrombin III; ACT/αAT, chimera carrying the reactive-site loop of α1-antitrypsin; ACT/PN1 chimera carrying the reactive-site loop of protease nexin 1; elastase, human leukocyte elastase; SI, stoichiometry of inhibition; pNA,p-nitroanilide. are protease inhibitors that are involved in the regulation of numerous protease cascades (e.g. blood coagulation and complement activation); they consist of 400–450 amino acids organized into three β-sheets and eight or nine α-helices connected by surface loops (1Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar). The connection between the A and C β-sheets constitutes the reactive-site loop. Although the precise inhibitory mechanism of serpins remains to be elucidated, all proposed models involve an initial interaction between the reactive-site loop of the serpin and the catalytic groove of the protease. Within the reactive-site loop, a P1residue 2Residues within the reactive-site loop are numbered by analogy with substrates as follows: P n … P3-P2-P1-P1′-P2′-P3′ … P n ′, where cleavage would occur at the P1–P1′ bond in a substrate. plays a crucial role in determining serpin specificity (2Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (833) Google Scholar, 3Bode W. Huber R. Eur. J. Biochem. 1992; 204: 433-451Crossref PubMed Scopus (1006) Google Scholar). However, many serpins have an arginine for P1 residue, yet inhibit different targets; thus other residues modulate the inhibitor function (4Owen M.C. Brennan S.O. Lewis J.H. Carrell R.W. N. Engl. J. Med. 1983; 309: 694-698Crossref PubMed Scopus (323) Google Scholar, 5Rubin H. Wang Z.m. Nickbarg E.B. McLarney S. Naidoo N. Schoenberger O.L. Johnson J.L. Cooperman B.S. J. Biol. Chem. 1990; 265: 1199-1207Abstract Full Text PDF PubMed Google Scholar, 6Patston P.A. Roodi N. Schifferli J.A. Bischoff R. Courtney M. Schapira M. J. Biol. Chem. 1990; 265: 10786-10791Abstract Full Text PDF PubMed Google Scholar, 7Keijer J. Ehrlich H.J. Linders M. Preissner K.T. Pannekoek H. J. Biol. Chem. 1991; 266: 10700-10707Abstract Full Text PDF PubMed Google Scholar, 8Owen M.C. George P.M. Lane D.A. Boswell D.R. FEBS Lett. 1991; 280: 216-220Crossref PubMed Scopus (16) Google Scholar, 9Sherman P.M. Lawrence D.A. Yang A.Y. Vandenberg E.T. Paielli D. Olson S.T. Shore J.D. Ginsburg D. J. Biol. Chem. 1992; 267: 7588-7595Abstract Full Text PDF PubMed Google Scholar, 10Björk I. Ylinenjärvi K. Olson S.T. Bock P.E. J. Biol. Chem. 1992; 267: 1976-1982Abstract Full Text PDF PubMed Google Scholar, 11Eldering E. Huijbregts C.C.M. Lubbers Y.T.P. Longstaff C. Hack C.E. J. Biol. Chem. 1992; 267: 7013-7020Abstract Full Text PDF PubMed Google Scholar, 12Phillips J.E. Cooper S.T. Potter E.E. Church F.C. J. Biol. Chem. 1994; 269: 16696-16700Abstract Full Text PDF PubMed Google Scholar, 13Engh R.A. Huber R. Bode W. Schulze A.J. Trends Biotechnol. 1995; 13: 503-510Abstract Full Text PDF PubMed Scopus (73) Google Scholar). The serpin α1-antichymotrypsin (ACT) is an acute phase plasma serpin (14Travis J. Salvesen G.S. Annu. Rev. Biochem. 1983; 52: 655-709Crossref PubMed Scopus (1483) Google Scholar, 15Baumann U. Huber R. Bode W. Grosse D. Lesjak M. Laurell C.B. J. Mol. Biol. 1991; 218: 595-606Crossref PubMed Scopus (157) Google Scholar, 16Wei A. Rubin H. Cooperman B.S. Christianson D.W. Nat. Struct. Biol. 1994; 1: 251-258Crossref PubMed Scopus (167) Google Scholar), which shares 45% sequence identity with α1-antitrypsin, 33% with antithrombin III, and 27% with protease nexin 1 (3Bode W. Huber R. Eur. J. Biochem. 1992; 204: 433-451Crossref PubMed Scopus (1006) Google Scholar, 17Chandra T. Stackhouse R. Kidd V.J. Robson K.J.H. Woo S.L.C. Biochemistry. 1983; 22: 5055-5061Crossref PubMed Scopus (162) Google Scholar, 18Gloor S. Odink K. Guenther J. Nick H. Monard D. Cell. 1986; 47: 687-693Abstract Full Text PDF PubMed Scopus (229) Google Scholar). ACT has Leu358 for P1 residue and inhibits chymotrypsin, pancreatic elastase, cathepsin G, and mast cell chymase (19Schechter N.M. Jordan L.M. James A.M. Cooperman B.S. Wang Z.m. Rubin H. J. Biol. Chem. 1993; 268: 23626-23633Abstract Full Text PDF PubMed Google Scholar, 20Cooperman B.S. Stavridi E. Nickbarg E. Rescorla E. Schechter N.M. Rubin H. J. Biol. Chem. 1993; 268: 23616-23625Abstract Full Text PDF PubMed Google Scholar). ACT does not inhibit trypsin-like enzymes, but single replacement for arginine of Leu358 dramatically alters its specificity: ACT(P1=Arg) inhibits trypsin and thrombin but not the elastase-like enzymes (5Rubin H. Wang Z.m. Nickbarg E.B. McLarney S. Naidoo N. Schoenberger O.L. Johnson J.L. Cooperman B.S. J. Biol. Chem. 1990; 265: 1199-1207Abstract Full Text PDF PubMed Google Scholar). The serpin α1-antitrypsin (also called α1-protease inhibitor; Refs. 2Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (833) Google Scholar and 14Travis J. Salvesen G.S. Annu. Rev. Biochem. 1983; 52: 655-709Crossref PubMed Scopus (1483) Google Scholar) inhibits several elastase-, chymotrypsin-, and trypsin-like enzymes, but substitution for arginine of its P1 methionine is still necessary for effective neutralization of thrombin, factor Xa, activated protein C, and urokinase (6Patston P.A. Roodi N. Schifferli J.A. Bischoff R. Courtney M. Schapira M. J. Biol. Chem. 1990; 265: 10786-10791Abstract Full Text PDF PubMed Google Scholar, 21Vidaud D. Emmerich J. Alhenc-Gelas M. Yvart J. Fiessinger J.N. Aiach M. J. Clin. Invest. 1992; 89: 1537-1543Crossref PubMed Scopus (28) Google Scholar, 22Hermans J.M. Stone S.R. Biochem. J. 1993; 295: 239-245Crossref PubMed Scopus (52) Google Scholar). Antithrombin III (14Travis J. Salvesen G.S. Annu. Rev. Biochem. 1983; 52: 655-709Crossref PubMed Scopus (1483) Google Scholar) has a P1arginine; it inhibits trypsin, thrombin, and factor Xa but not activated protein C (23Stone S.R. Hanspeter N. Hofsteenge J. Monard D. Arch. Biochem. Biophys. 1987; 252: 237-244Crossref PubMed Scopus (92) Google Scholar, 24Pratt C.W. Whinna H.C. Church F.C. J. Biol. Chem. 1992; 267: 8795-8801Abstract Full Text PDF PubMed Google Scholar, 25Stone S.R. Brown-Luedi M.L. Rovelli G. Guidolin A. McGlynn E. Monard D. Biochemistry. 1994; 33: 7731-7735Crossref PubMed Scopus (52) Google Scholar). Finally, protease nexin 1, which also has a P1 arginine, rapidly inactivates thrombin, urokinase, trypsin, and activated protein C (18Gloor S. Odink K. Guenther J. Nick H. Monard D. Cell. 1986; 47: 687-693Abstract Full Text PDF PubMed Scopus (229) Google Scholar, 22Hermans J.M. Stone S.R. Biochem. J. 1993; 295: 239-245Crossref PubMed Scopus (52) Google Scholar, 23Stone S.R. Hanspeter N. Hofsteenge J. Monard D. Arch. Biochem. Biophys. 1987; 252: 237-244Crossref PubMed Scopus (92) Google Scholar). To document further the role of the reactive site loop in controlling the specificity, we have prepared and characterized various chimeras, using ACT as a framework to carry the loop of other serpins with overlapping specificities. Results suggest that, in addition to the sequence of the reactive site loop, the specificity of serpins originates from secondary binding sites and conformational constraints. Human activated protein C was a generous gift from Drs. J. Stenflo and A. Öhlin (University of Lund, Malmö, Sweden). Human leukocyte elastase (elastase) was from Athens Inc. (Athens, GA). Bovine pancreatic trypsin (l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated) and bovine chymotrypsin (1-chloro-3-tosylamido-7-amino-2-heptanone-treated) were purchased from Worthington (Lorne Laboratories, Twyford, UK), and human urokinase (low molecular weight) was from Calbiochem (Novobiochem, Nottingham, UK). Human thrombin and bovine factor Xa were prepared as described previously (26Stone S.R. Hofsteenge J. Biochemistry. 1986; 25: 4622-4628Crossref PubMed Scopus (514) Google Scholar, 27Le Bonniec B.F. Guinto E.R. Esmon C.T. J. Biol. Chem. 1992; 267: 6970-6976Abstract Full Text PDF PubMed Google Scholar). Mutants of ACT were produced and purified essentially according to Rubin et al. (5Rubin H. Wang Z.m. Nickbarg E.B. McLarney S. Naidoo N. Schoenberger O.L. Johnson J.L. Cooperman B.S. J. Biol. Chem. 1990; 265: 1199-1207Abstract Full Text PDF PubMed Google Scholar, 28Rubin H. Plotnick M. Wang Z.-M. Liu X. Zhong Q. Schecter N.M. Cooperman B.S. Biochemistry. 1994; 33: 7627-7633Crossref PubMed Scopus (53) Google Scholar). Reactive-site loops were exchanged by cassette mutagenesis using the expression vector pACT generously given by Dr. H. Rubin (University of Pennsylvania, Philadelphia, PA), in which two restriction sites (KpnI and MluI) had been engineered within the ACT coding sequence. The engineered KpnI site, between the codon for the P9 and P10 residues, results in the replacement of the alanines of wild-type ACT with glycine and threonine; the MluI site, between the codons for the P10′ and P11′ residues, exchanges the P10′ valine to threonine (28Rubin H. Plotnick M. Wang Z.-M. Liu X. Zhong Q. Schecter N.M. Cooperman B.S. Biochemistry. 1994; 33: 7627-7633Crossref PubMed Scopus (53) Google Scholar, 29Kilpatrick L. Johnson J.L. Nickbarg E.B. Wang Z.M. Clifford T.F. Banach M. Cooperman B.S. Douglas S.D. Rubin H. J. Immunol. 1991; 146: 2388-2393PubMed Google Scholar). To express chimeras ACT/ATIII, ACT/αAT, ACT/αAT(P1=Arg) and ACT/PN1 (TableI), oligonucleotides flanked by the appropriate restriction sites and encoding the reactive-site loops of antithrombin III (5′-C-ACT-GCT-GTT-GTA-ATC-GCT-GGT-CGT-TCT-CTG-AAC-CCG-AAC-CGT-GTT-ACC-A-3′ and 3′-CA-TGG-TGA-CGA-CAA-CAT-TAG-CGA-CCA-GCA-AGA-GAC-TTG-GGC-TTG-GCA-CAA-TGG-TGC-GC-5′), of α1-antitrypsin (5′-C-ATG-TTC-CTG-GAA-GCT-ATC-CCG-ATG-AGC-ATC-CCG-CCA-GAA-A-3′ and 3′-CA-TGG-TAC-AAG-GAC-CTT-CGA-TAG-GGC-TAC-TCG-TAG-GGC-GGT-CTT-TGC-GC-5′), of α1-antitrypsin(P1=Arg) (same as above with the second ATG codon replaced by CGT), or of protease nexin 1 (5′-C-ACA-ACT-GCA-ATT-CTC-ATT-GCA-AGA-TCA-TCG-CCT-CCC-TGG-A-3′ and 3′-CA-TGG-TGT-TGA-CGT-TAA-GAG-TAA-CGT-TCT-AGT-AGC-GGA-GGG-ACC-TGC-GC-5′) were ligated in pACT by standard techniques (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Chimera ACT(des-TIVR), in which the reactive-site loop of ACT was shortened by four residues (Thr366, Ile367, Val368, and Arg369), was prepared by removing the corresponding codons from pACT and reconstruction of anMluI site between the codons for the P6′ and P7′ residues. Briefly, pACT was used as a template, with oligonucleotides 5′-GAC-CCC-CAA-GAT-ACT-CAT-CAG-3′ and 3′-A-AAC-CAA-CTT-TGC-GCA-TGG-TAG-CAT-GC-5′ as forward/reverse primers in a first polymerase chain reaction, and oligonucleotides 5′-T-TTG-GTT-GAA-ACG-CGT-ACC-ATC-GTA-CG-3′ and 3′-CGG-AAG-TTG-GGT-CAG-TCG-AGG-AAG-5′ as forward/reverse primers in a second polymerase chain reaction. The resulting fragments (517 and 624 base pairs) were digested with MluI, ligated with T4 DNA ligase, and utilized as template for a third amplification, using as primers the forward of the first and the reverse of the second polymerase chain reactions. The resulting 1124-base pair fragment was cloned into pCRII vector (Invitrogen, Abingdon, UK), according to the manufacturer's instruction. The pACT(des-TIVR) expression vector with a “short” reactive-site loop was prepared by exchanging the KpnI/SphI fragment of pACT for the corresponding 618-base pair KpnI/SphI fragment of pCRII. The resulting expression vector pACT(des-TIVR) was used for preparation of ACT(des-TIVR) and to construct, by cassette mutagenesis, the expression vectors for ACT/αAT(des-TIVR), ACT/αAT(P1=Arg; des-TIVR), and ACT/PN1(des-TIVR). Recombinants ACT variants were purified in mg quantities by a combination of anion-exchange and DNA-cellulose chromatography, as described previously (30Djie M.Z. Le Bonniec B.F. Hopkins P.C.R. Hipler K. Stone S.R. Biochemistry. 1996; 35 (2nd Ed.): 11461-11469Crossref PubMed Scopus (40) Google Scholar). Final products appeared homogeneous by Coomassie Blue staining, following SDS-polyacrylamide gel electrophoresis.Table IIk on values for various proteases of the chimeras having a P 1 argininek on (m−1s−1)TrypsinThrombinFXaAPCUrokinaseACTP1=Arg)4.1 × 1051.8 × 1032.1 × 1011.9 × 102<10Antithrombin III1.4 × 1051.1 × 1043.1 × 103<10<10ACT/ATIII3.7 × 1051.4 × 1037.0 × 103<10<10ACT(P1=Arg; P2=Gly)5.5 × 1051.5 × 1033.0 × 103<10NDProtease nexin 14.7 × 1062.1 × 1066.5 × 1035.2 × 1032.9 × 105ACT/PN11.8 × 1052.4 × 1031.4 × 1031.4 × 101<10ACT/PN1(des-TIVR)2.7 × 1044.2 × 1022.5 × 103<10<10ACT(P1=Arg; P2=Ala)4.2 × 1056.3 × 1026.0 × 1027.8 × 101NDα1-Antitrypsin(P1=Arg)4.2 × 1053.1 × 1051.9 × 1047.0 × 1018.9 × 103ACT/αAT(P1=Arg)2.6 × 1064.9 × 1039.0 × 1014.6 × 101<10ACT/αAT(P1=Arg; des-TIVR)3.2 × 1048.6 × 1022.0 × 101<10<10ACT(P1=Arg; P2=Pro)4.4 × 1053.3 × 1046.8 × 1022.0 × 102NDEach slow-binding inhibition experiment was performed at least twice; the k on value given represents the weighted mean of these determinations. The standard errors of the weighted means were 5% or less of the mean value. Abbreviations used are: FXa, factor Xa; APC, activated protein C; ND. not determined. Values of the ACT double mutants (P1 arginine and either P2 proline, alanine or glycine) were taken from Djie et al. (30Djie M.Z. Le Bonniec B.F. Hopkins P.C.R. Hipler K. Stone S.R. Biochemistry. 1996; 35 (2nd Ed.): 11461-11469Crossref PubMed Scopus (40) Google Scholar). Open table in a new tab Table INomenclature of the ACT mutants, sequence of their reactive-site loop, and stoichiometry of inhibition (SI)ACT variantReactive-site loop sequenceSIP10 P1–P1′ P13′ChymotrypsinElastaseACT ACTAATAVKITLL–SALVETRTIVRFN3.4100ACT(des-TIVR)AATAVKITLL–SALVETR....FN3.3NDACT/αATGTMFLEAIPM–SIPPETRTIVRFN1.8111ACT/αAT(des-TIVR)GTMFLEAIPM–SIPPETR....FN3.160P10 P1–P1′ P13′TrypsinThrombinACT(P1=Arg)AATAVKITLR–SALVETRTIVRFN3.01.8ACT(P1=Arg; P2=Pro)AATAVKITPR–SALVETRTIVRFN1062.9ACT/αAT(P1=Arg)GTMFLEAIPRSIPPETRTIVRFN1523.5ACT/αAT(P1=Arg; des-TIVR)GTMFLEAIPR–SIPPETR....FN62510.0ACT(P1=Arg; P2=Ala)AATAVKITAR–SALVETRTIVRFN1.33.3ACT/PN1GTTTAILIAR–SSPPWTRTIVRFN1.43.5ACT/PN1(des-TIVR)GTTTAILIAR–SSPPWTR....FN1.93.3ACT(P1=Arg; P2=Gly)AATAVKITGR–SALVETRTIVRFN1.92.3ACT/ATIIIGTTAVVIAGR–SLNPNRVTT.RFN7.11.6Comparison with ACT of the P10–P13′ residues of the various serpins and chimeras used in this study. Amino acids that differ from ACT are underlined; dot indicates that the corresponding residue is absent. SI value represents the number of moles of serpin required to inhibit 1 mol of enzyme; its estimation was not reliable (ND) for elastase inhibition by ACT(des-TIVR). SI values of the ACT double mutants (P1 arginine and either P2 proline, alanine, or glycine) were taken from Djie et al. (30Djie M.Z. Le Bonniec B.F. Hopkins P.C.R. Hipler K. Stone S.R. Biochemistry. 1996; 35 (2nd Ed.): 11461-11469Crossref PubMed Scopus (40) Google Scholar). Open table in a new tab Each slow-binding inhibition experiment was performed at least twice; the k on value given represents the weighted mean of these determinations. The standard errors of the weighted means were 5% or less of the mean value. Abbreviations used are: FXa, factor Xa; APC, activated protein C; ND. not determined. Values of the ACT double mutants (P1 arginine and either P2 proline, alanine or glycine) were taken from Djie et al. (30Djie M.Z. Le Bonniec B.F. Hopkins P.C.R. Hipler K. Stone S.R. Biochemistry. 1996; 35 (2nd Ed.): 11461-11469Crossref PubMed Scopus (40) Google Scholar). Comparison with ACT of the P10–P13′ residues of the various serpins and chimeras used in this study. Amino acids that differ from ACT are underlined; dot indicates that the corresponding residue is absent. SI value represents the number of moles of serpin required to inhibit 1 mol of enzyme; its estimation was not reliable (ND) for elastase inhibition by ACT(des-TIVR). SI values of the ACT double mutants (P1 arginine and either P2 proline, alanine, or glycine) were taken from Djie et al. (30Djie M.Z. Le Bonniec B.F. Hopkins P.C.R. Hipler K. Stone S.R. Biochemistry. 1996; 35 (2nd Ed.): 11461-11469Crossref PubMed Scopus (40) Google Scholar). Kinetics were performed in 50 mm Tris-HCl, pH 7.8, containing 0.1 m NaCl, 0.2% poly(ethylene glycol) M r 6000, and 1 mg/ml bovine serum albumin (protease-free, Sigma). Assays with activated protein C contained in addition 5 mm CaCl2. The chromogenic substrates S-2222 (benzyl-CO-Ile-Glu-(γ-OR)-Gly-Arg-pNA, where R is H or CH3, and pNA indicatesp-nitroanilide) was used with factor Xa (K m = 547 μm). S-2238 (H-d-Phe-Pip-Arg-pNA) (Pip indicatesl-pipecolyl) was used with thrombin and activated protein C (K m = 3.6 and 315 μm, respectively), as well as S-2266 (H-d-Val-Leu-Arg-pNA;K m = 486 and 221 μm, respectively). S-2288 (H-d-Ile-Pro-Arg-pNA) and S-2302 (H-d-Pro-Phe-Arg-pNA) were used with trypsin (K m = 19 and 134 μm, respectively). S-2444 (<Glu-Gly-Arg-pNA) was used with urokinase (K m = 90 μm). The above peptidyl-pNA substrates were purchased from Chromogenix (Mölndal, Sweden). Succinyl-Ala-Ala-Pro-Phe-pNA was used with chymotrypsin (K m = 53 μm), and N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA was used with elastase (K m = 121 μm); both were purchased from Sigma. Substrate concentrations were determined from their absorbance at 342 nm using a molar extinction coefficient of 8270 cm−1 (32Lottenberg R. Hall J.A. Blinder M. Binder E.P. Jackson C.M. Biochim. Biophys. Acta. 1983; 742: 539-557Crossref PubMed Scopus (114) Google Scholar). Choice of the pNA substrate depended whether competition between hydrolysis and inhibition was desired; substrates with low K m values were used to increase the apparent half-life of complex formation, and those with high K m values were used to minimize competition. Stoichiometry of inhibition (SI, i.e. the number of moles of serpin required to inhibit 1 mol of protease) was determined as described previously (30Djie M.Z. Le Bonniec B.F. Hopkins P.C.R. Hipler K. Stone S.R. Biochemistry. 1996; 35 (2nd Ed.): 11461-11469Crossref PubMed Scopus (40) Google Scholar), using chymotrypsin and elastase for the chimeras having a P1 leucine or methionine and using trypsin and thrombin for the chimeras having a P1 arginine. The ability of the chimeras to form SDS-stable complexes was examined by incubating the variant (2 μm) with the protease (1 μm) in 50 mm Tris-HCl buffer, pH 7.8, containing 100 mm NaCl, and 0.2% (w/v) poly(ethylene glycol)M r 6000 for 30 min at room temperature. The sample was denatured at 65 °C for 10 min in 0.37 mTris-HCl, pH 8.8, containing 1% SDS (w/v), 10% glycerol (v/v), and 5% β-mercaptoethanol (v/v) and analyzed by SDS-polyacrylamide gel electrophoresis (gradient 10–20% acrylamide). The overall association rate constant for the formation of a protease-serpin complex (k on) was estimated by analysis of data from progress curve kinetics completed with a large excess of serpin (6 concentrations, between 0.32 and 10 μm) over the enzyme (a single concentration between 0.01 and 1 nm, depending upon the protease). Inhibition reactions were followed for up to 3 h using a Hewlett-Packard diode array spectrophotometer, but only data corresponding to less than 10% substrate hydrolysis were analyzed. Reactions were initiated by addition of the enzyme at a concentration such that the velocity in the absence of inhibitor was about 0.2 μm min−1 with the appropriate peptidyl-substrate (100 μm). Data were analyzed according to the equation for slow binding inhibition to yield estimates fork on as described previously (30Djie M.Z. Le Bonniec B.F. Hopkins P.C.R. Hipler K. Stone S.R. Biochemistry. 1996; 35 (2nd Ed.): 11461-11469Crossref PubMed Scopus (40) Google Scholar). We have constructed ACT chimeras in which the length of the reactive-site loop corresponds to that of either the donor or acceptor (ACT/αAT and ACT/PN1 carrying long reactive-site loop, as well as ACT/αAT(des-TIVR) and ACT/PN1(des-TIVR)carrying shorter loop); because the reactive-site loop of antithrombin III is only one amino acid shorter than that of ACT, only the ACT/ATIII chimera was constructed (Table I). The selectivity and effectiveness of each chimera toward a number of possible targets were evaluated using three criteria: SI value, ability to form an SDS-stable complex, and k on value. Chimeras with proline in P2 and arginine in P1exhibited SI values higher than 150 with trypsin and lower than 10 with thrombin (Table I). This is consistent with our observation (30Djie M.Z. Le Bonniec B.F. Hopkins P.C.R. Hipler K. Stone S.R. Biochemistry. 1996; 35 (2nd Ed.): 11461-11469Crossref PubMed Scopus (40) Google Scholar) that replacement of the P2 leucine of ACT(P1=Arg)with proline causes a dramatic increase of the SI value with trypsin but not with thrombin. When P1 was either methionine or leucine, SI values were higher than 60 with elastase and lower than 3 with chymotrypsin (regardless of the P2 residue). When the loop of the ACT/αAT chimeras was shorter, SI values were higher for chymotrypsin, trypsin, and thrombin inhibition; the opposite was true for elastase. In contrast, the length of loop did not change the SI value with the ACT/PN1 chimeras. Thus SI value depended upon the sequence and the length of the reactive-site loop, as well as upon the target considered. Attempts to detect SDS-stable complex formation followed incubation of a slight excess of serpin with μm quantities of the potential target (i.e. in conditions where cleavage reaction occurs substantially). Three patterns were observed (Fig.1) as follows: 1) the formation of SDS-stable complex, but the presence of intermediate size fragments attributable to their degradation by remaining (active) protease (38Stavridi E.S. O'Malley K. Lukacs C.M. Moore W.T. Lambris J.D. Christianson D.W. Rubin H. Cooperman B.S. Biochemistry. 1996; 35: 10608-10615Crossref PubMed Scopus (75) Google Scholar); 2) absence of detectable complex, but accumulation of material migrating as ACT having the reactive-site loop cleaved; and 3) absence of detectable reaction (neither complex formation nor cleavage). Chimeras with leucine or methionine in P1 position all formed an SDS-stable complex with chymotrypsin (pattern 1), whereas consistent with the high SI value, chimeras were mainly cleaved following incubation with elastase (pattern 2). Chimeras having arginine in P1 position exhibited pattern 1 with thrombin, whereas the pattern was either 1 or 2 following incubation with trypsin (again, consistent with the high SI value, pattern was 2 with proline in P2, and pattern 1 was otherwise). Regardless of the chimera, there was no clear evidence of SDS-stable complex formation nor of cleavage reaction with factor Xa, activated protein C, and urokinase; the predominant bands were those of the intact proteins (pattern 3). Finally, ACT/ATIII exhibited a mixed pattern. Thus, with the exception of trypsin, the pattern was specific for the target rather than for the chimera. Under the conditions of the slow binding assays (low enzyme concentration and very large excess of serpin), depletion of the inhibitor by the cleavage reaction becomes negligible, as the total amount of cleaved inhibitor depends on the absolute amount of enzyme (30Djie M.Z. Le Bonniec B.F. Hopkins P.C.R. Hipler K. Stone S.R. Biochemistry. 1996; 35 (2nd Ed.): 11461-11469Crossref PubMed Scopus (40) Google Scholar, 34Waley S.G. Biochem. J. 1985; 227: 843-849Crossref PubMed Scopus (158) Google Scholar, 35Stone S.R. Hermans J.M. Biochemistry. 1995; 34: 5164-5172Crossref PubMed Scopus (41) Google Scholar). The k on values obtained (TableII) did not reveal a clear relationship between the nature of the reactive-site loop and the specificity or function of the chimera, but in two instances only, the k onvalues were higher when length of the reactive-site loop was shorter than that of ACT. Substitution of the reactive-site loop of ACT with that of antithrombin III transposed quite effectively the specificity of antithrombin III to ACT (TableII). ACT/ATIII exhibited k on values within 10-fold those of antithrombin III for trypsin, thrombin, and factor Xa inhibition, whereas k on values were lower than 10 m−1 s−1 with activated protein C and urokinase. Thus, ACT/ATIII exhibited a selectivity resembling that of antithrombin III. However, this apparent success must be tempered by the observation that simple substitution of the P1 leucine with arginine largely accounts for the inhibitory properties of ACT/ATIII with trypsin or thrombin, whereas further substitution of the P2 leucine with glycine is sufficient to mimic antithrombin III with every protease (30Djie M.Z. Le Bonniec B.F. Hopkins P.C.R. Hipler K. Stone S.R. Biochemistry. 1996; 35 (2nd Ed.): 11461-11469Crossref PubMed Scopus (40) Google Scholar). Thus, substitution of the whole reactive-site loop did not alter the selectivity of the ACT mutant having the P1 and P2 residues of antithrombin III. Except for factor Xa inhibition, introducing the reactive-site loop of protease nexin 1 into ACT did not result in an appropriate transfer of specificity (TableII). Failure was most evident with urokinase;k on values were at least 4 orders of magnitude lower than with protease nexin 1. In fact, regardless of the reactive-site loop, none of the ACT variants neutralized urokinase. The lack of urokinase inhibition was due to an absence of reaction rather than to the ACT variants acting as substrates; no cleavage reaction was detected by polyacrylamide gel electrophoresis. Thek on values for thrombin inhibition were also dramatically lower than with the loop donor (875- and 5000-fold with ACT/PN1 and ACT/PN1(des-TIVR), respectively), and activated protein C inhibition was hardly detectable. However, the reactive-site loop of protease nexin 1 was functional in the context of the ACT framework. Less than 5-fold separated the k onvalues of factor Xa inhibition by protease nexin 1 and ACT/PN1 and less than 27-fold separated those for trypsin inhibition. Factor Xa was also a remarkable exception, because shortening the loop increased the inhibitory act" @default.
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• W2091001049 title "Intrinsic Specificity of the Reactive Site Loop of α1-Antitrypsin, α1-Antichymotrypsin, Antithrombin III, and Protease Nexin I" @default.
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