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• W2043908422 abstract "Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 ± 2 μm) and aprotinin (Ki 1.13 ± 0.07 μm) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 ± 32 nm) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 ± 0.52 μm) was non-competitive, whereas inhibition by active site-inhibited factor IXaβ was competitive (Ki 0.33 ± 0.05 μm). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 ± 14 μm) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions. Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 ± 2 μm) and aprotinin (Ki 1.13 ± 0.07 μm) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 ± 32 nm) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 ± 0.52 μm) was non-competitive, whereas inhibition by active site-inhibited factor IXaβ was competitive (Ki 0.33 ± 0.05 μm). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 ± 14 μm) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions. Factor IX (fIX) 1The abbreviations used are: fIX, factor IX; fIXaβ, factor IXaβ; fIXai, active site-inhibited factor IXaβ; fXI, factor XI; fXIa, factor XIa; fXIaCD, factor XIa catalytic domain; A, apple domain; TF, tissue factor, pAB, p-aminobenzamidine; PK, prekallikrein. is the zymogen precursor of a trypsin-like plasma protease, factor IXaβ (fIXaβ), that contributes to fibrin clot formation through proteolytic activation of factor X (1Furie B. Furie B. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (995) Google Scholar, 2Davie E. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1635) Google Scholar). fIX conversion to fIXaβ is achieved through two proteolytic cleavages after Arg145 and Arg180, releasing an 11-kDa activation peptide (3Fujikawa K. Lagaz M. Kato H. Davie E. Biochemistry. 1974; 13: 4508-4516Crossref PubMed Scopus (129) Google Scholar, 4DiScipio R. Kurachi K. Davie E. J. Clin. Investig. 1978; 61: 1528-1538Crossref PubMed Scopus (178) Google Scholar, 5Lindquist P. Fujikawa K. Davie E. J. Biol. Chem. 1978; 253: 1902-1909Abstract Full Text PDF PubMed Google Scholar). During hemostasis, fIX activation occurs through two distinct pathways mediated by the protease factors VIIa and XIa (fXIa) (3Fujikawa K. Lagaz M. Kato H. Davie E. Biochemistry. 1974; 13: 4508-4516Crossref PubMed Scopus (129) Google Scholar, 4DiScipio R. Kurachi K. Davie E. J. Clin. Investig. 1978; 61: 1528-1538Crossref PubMed Scopus (178) Google Scholar, 6Osterud B. Rapaport S. Proc. Natl. Acad. Sci. (U. S. A.). 1977; 74: 5260-5264Crossref PubMed Scopus (598) Google Scholar, 7Osterud B. Bouma B. Griffin G. J. Biol. Chem. 1978; 253: 5946-5951Abstract Full Text PDF PubMed Google Scholar, 8Broze G. Girard T. Novotny W. Biochemistry. 1990; 29: 7539-7546Crossref PubMed Scopus (309) Google Scholar). Plasma coagulation is initiated when factor VIIa binds to the integral membrane protein tissue factor (TF) at a wound site (8Broze G. Girard T. Novotny W. Biochemistry. 1990; 29: 7539-7546Crossref PubMed Scopus (309) Google Scholar, 9Rapaport S. Rao L. Arterioscler. Thromb. 1992; 12: 1111-1121Crossref PubMed Scopus (272) Google Scholar, 10Nemerson Y. Semin. Hematol. 1992; 29: 170-176PubMed Google Scholar). fIXaβ generated by factor VIIa/TF converts factor X to Xa and is probably involved in initial fibrin formation and sustained thrombin production. Activation of fIX by fXIa likely occurs after initial fibrin formation (11Broze G. Semin. Hematol. 1992; 29: 159-169PubMed Google Scholar, 12Walsh P. Semin. Hematol. 1992; 29: 189-201PubMed Google Scholar) and is required for maintenance of clot stability, particularly in tissues rich in fibrinolytic activity that would otherwise quickly degrade the clot (13Asakai R. Chung D. Davie E. Seligsohn U. N. Eng. J. Med. 1991; 325: 153-158Crossref PubMed Scopus (322) Google Scholar, 14Seligsohn U. Thromb. Haemostasis. 1993; 70: 68-70Crossref PubMed Scopus (127) Google Scholar). fXIa differs from other coagulation proteases in several important aspects. fXI is a homodimer (15Bouma B. Griffin J. J. Biol. Chem. 1977; 252: 6432-6437Abstract Full Text PDF PubMed Google Scholar, 16McMullen B. Fujikawa K. Davie E. Biochemistry. 1991; 30: 2056-2060Crossref PubMed Scopus (122) Google Scholar), whereas other coagulation proteases are monomers (2Davie E. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1635) Google Scholar). Although the C-terminal portion of the fXI polypeptide is a typical trypsin-like protease domain, the N-terminal non-catalytic region contains four repeats called apple domains (A1–A4 from the N terminus) not found on other coagulation proteases (16McMullen B. Fujikawa K. Davie E. Biochemistry. 1991; 30: 2056-2060Crossref PubMed Scopus (122) Google Scholar, 17Fujikawa K. Chung D. Hendrickson L. Davie E. Biochemistry. 1986; 25: 2417-2424Crossref PubMed Scopus (204) Google Scholar). Furthermore, fXIa lacks the phospholipid-binding Gla domain characteristic of vitamin K-dependent coagulation proteases. Indeed, although phospholipid lowers Km for most coagulation protease reactions several orders of magnitude, it has little effect on fIX activation by fXIa (4DiScipio R. Kurachi K. Davie E. J. Clin. Investig. 1978; 61: 1528-1538Crossref PubMed Scopus (178) Google Scholar, 7Osterud B. Bouma B. Griffin G. J. Biol. Chem. 1978; 253: 5946-5951Abstract Full Text PDF PubMed Google Scholar, 18Mannhalter C. Schiffman S. Deutsch E. Br. J. Haematol. 1984; 56: 261-271Crossref PubMed Scopus (14) Google Scholar). The molecular mechanism by which fXIa activates fIX is not completely understood, and the unusual structural features of fXIa cited above make it difficult to extrapolate from data obtained for other coagulation proteases. Enzymes involved in fibrin formation are members of the chymotrypsin family of serine proteases (2Davie E. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1635) Google Scholar, 19Neurath H. Science. 1984; 224: 350-357Crossref PubMed Scopus (403) Google Scholar, 20Bode W. Brandstetter H. Mather T. Stubbs M. Thromb. Haemostasis. 1997; 78: 501-511Crossref PubMed Scopus (99) Google Scholar). Despite having relatively similar catalytic domains, these enzymes exhibit specific substrate recognition (21Mann K. Jenny R. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (452) Google Scholar). Substrate specificity and affinity for many serine proteases involved in digestive or degradative processes are governed primarily by interactions between the protease catalytic domain and sites on the substrate near the protease cleavage site (22Perona J. Craik C. Protein Sci. 1995; 4: 337-360Crossref PubMed Scopus (765) Google Scholar, 23Perona J. Craik C. J. Biol. Chem. 1997; 272: 29987-29990Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 24Krem M. Rose T. Di Cera E. J. Biol. Chem. 1999; 274: 28063-28066Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These interactions, which involve active site groups (primarily the S1–S3 substrate-binding subsites) and surface loops on the catalytic domain, are critical for proper alignment of substrate with the active site. More specialized proteases, including those involved in coagulation, frequently have domains outside of the protease domain that are required for proper function (1Furie B. Furie B. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (995) Google Scholar, 2Davie E. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1635) Google Scholar, 25Doolittle R. Feng D. Cold Spring Harbor Symp. Quant. Biol. 1987; 52: 869-874Crossref PubMed Scopus (60) Google Scholar). A substantial body of evidence obtained by structural biology approaches indicates that binding interactions outside the protease domain are important determinants of substrate affinity and specificity in coagulation reactions (26Duffy E. Parker E. Mutucumarana V. Johnson A. Lollar J. J. Biol. Chem. 1992; 267: 17006-17011Abstract Full Text PDF PubMed Google Scholar, 27Shobe J. Dickinson C. Edgington T. Ruff W. J. Biol. Chem. 1999; 274: 24171-24175Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 28Baugh R. Dickinson C. Ruf W. Krishnaswamy S. J. Biol. Chem. 2000; 275: 28826-28833Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 29Gale A. Tsavaler A. Griffin J. J. Biol. Chem. 2002; 277: 28836-28840Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 30Aktimur A. Gabriel M. Gailani D. Toomey J. J. Biol. Chem. 2003; 278: 7981-7987Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Studies of prothrombin (31Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (87) Google Scholar, 32Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 33Boskovic D. Krishnaswamy S. J. Biol. Chem. 2000; 275: 38561-38570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 34Orcutt S. Pietropaolo C. Krishnaswamy S. J. Biol. Chem. 2002; 277: 46191-46196Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 35Boskovic D. Troxler T. Krishnaswamy S. J. Biol. Chem. 2004; 279: 20786-20793Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) and factor X activation (28Baugh R. Dickinson C. Ruf W. Krishnaswamy S. J. Biol. Chem. 2000; 275: 28826-28833Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) identified a primary role for these exosite interactions in determining binding affinity and specificity and established experimental approaches for determining the functional importance of exosite interactions in formation of productive enzyme-substrate complexes. A two-step model of coagulation protease zymogen activation has been proposed in which substrate binds initially to an exosite followed by docking at the active site and subsequent catalysis (28Baugh R. Dickinson C. Ruf W. Krishnaswamy S. J. Biol. Chem. 2000; 275: 28826-28833Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 31Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (87) Google Scholar). Several lines of evidence suggest that fIX activation involves exosite interactions with the fXIa heavy chain. Km for fIX activation by the isolated fXIa catalytic domain is ~25-fold higher than for intact fXIa (36Sinha D. Seaman F. Walsh P. Biochemistry. 1987; 26: 3768-3775Crossref PubMed Scopus (51) Google Scholar). Recombinant fXIa in which the A3 domain is replaced with the homologous domain from plasma kallikrein also activates fIX with a substantially greater Km when compared with wild type fXIa (37Sun Y. Gailani D. J. Biol. Chem. 1996; 271: 29023-29028Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 38Sun M. Zhao M. Gailani D. J. Biol. Chem. 1999; 274: 36373-36378Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Although these studies clearly show that the fXIa heavy chain interacts with fIX, the importance of these interactions, relative to those at the protease active site, in productive substrate recognition is not clear. Here, we investigated the importance of exosite binding to substrate recognition of fIX by fXIa. Materials—S299 (methyl-sulfonyl-d-cyclo-hexyl-glycyl-glycyl-arginine-p-nitroanilide) was from American Diagnostics (Greenwich, CT), and S2366 (l-pyroglutamyl-l-prolyl-l-arginine-p-nitroanilide) was from DiaPharma (West Chester, OH). Aprotinin and p-aminobenzamidine (pAB) were from Sigma. Plasma Proteins—fIX was prepared from human plasma collected into acid-citrate-dextrose. Plasma (2 liters) at 4 °C was supplemented with benzamidine to 20 mm, and 160 ml of 1 m BaCl2 was slowly added with stirring. After 1 h, the precipitate was pelleted at 10,000 × g for 20 min, washed twice with 1 liter of 10 mm Tris-HCl, pH 7.5, 10 mm NaCl, 20 mm benzamidine, 10 mm BaCl2, and resuspended in 100 ml of 10 mm Tris-HCl, pH 7.5, 20 mm benzamidine, 20% saturated ammonium sulfate, 20 μg/ml soybean trypsin inhibitor. The suspension was dialyzed against 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 20 mm benzamidine, 5 mm EDTA and then against 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 20 mm benzamidine, 2.5 mm CaCl2. After centrifugation, fIX was purified from supernatant by antibody-affinity chromatography by using the calcium-dependent anti-human fIX monoclonal IgG SB 249417 (Dr. John Toomey, GlaxoSmithKline) linked to Affi-Gel-10 (Bio-Rad) (30Aktimur A. Gabriel M. Gailani D. Toomey J. J. Biol. Chem. 2003; 278: 7981-7987Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). After loading, the column was washed with 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 5 mm CaCl2, 5 mm benzamidine and then eluted with 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 25 mm EDTA, 5 mm benzamidine. Protein-containing fractions were concentrated by ultrafiltration and dialyzed against 50 mm Tris-HCl, pH 7.5, 100 mm NaCl (TBS). Purity was determined by SDS-PAGE and concentration by colorimetric assay (Bio-Rad). Human fXIa, fIXaβ, and fIXai (fIXaβ with the active site inhibited by Glu-Gly-Arg-chloromethyl ketone) were from Hematologic Technologies (Essex Junction, VT). Recombinant fXIa Catalytic Domain—The human fXI cDNA (17Fujikawa K. Chung D. Hendrickson L. Davie E. Biochemistry. 1986; 25: 2417-2424Crossref PubMed Scopus (204) Google Scholar) was altered by using a QuikChange mutagenesis kit (Stratagene, La Jolla, CA), converting the TGT triplet for Cys362 to TCT (Ser) and converting the TGC triplet for Cys482 to AGC (Ser). The construct (fXI-Ser362,482) codes for a protein lacking the disulfide bond that connects the fXIa catalytic domain to the heavy chain. fXI-Ser362,482 cDNA was ligated into vector pJVCMV (38Sun M. Zhao M. Gailani D. J. Biol. Chem. 1999; 274: 36373-36378Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), and 50 × 106 293 fibroblasts (ATCC CRL 1573) were cotransfected by electroporation (Electrocell Manipulator 600 BTX, San Diego, CA) with 40 μg of construct and 2 μg of pRSVneo (38Sun M. Zhao M. Gailani D. J. Biol. Chem. 1999; 274: 36373-36378Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Cells were grown in Dulbecco's modified Eagle's medium, 5% fetal bovine serum, 500 μg/ml G418. Supernatants from G418-resistant clones were tested by enzyme-linked immunosorbent assay by using goat anti-human fXI antibody (Affinity Biologicals, Hamilton, Ontario, Canada). Expressing clones were expanded in 175-cm2 flasks, and conditioned medium was collected every 48 h. fXI-Ser362,482 was purified from medium on an anti-fXI IgG 1G5 affinity column (38Sun M. Zhao M. Gailani D. J. Biol. Chem. 1999; 274: 36373-36378Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Purified fXI-Ser362,482 (~300 μg/ml) was activated with 5 μg/ml factor XIIa (Enzyme Research Laboratories, South Bend, IN) at 37 °C, and complete activation was confirmed by SDS-PAGE. Activated protein was reapplied to the 1G5 antibody column to separate the fXIa-Ser362,482 catalytic domain (fXIaCD), which binds to the column, from factor XIIa and the fXIa-Ser362,482 heavy chain, which flow through the column. fXIa Hydrolysis of S2366 —fXIa (6 nm active sites, 3 nm protein) or fXIaCD (6 nm active sites, 6 nm protein) was diluted in TBS containing 0.1 mg/ml bovine serum albumin (TBSA), 5 mm CaCl2, and S2366 (50–2000 μm) in the presence or absence of pAB, aprotinin, fIX, or fIXai. Rates of generation of free p-nitroaniline were measured by continuous monitoring of absorbance at 405 nm by using 100-μl reaction volumes (3-mm path length) in a SpectraMax 340 microtiter plate reader (Molecular Devices Corp., Sunnyvale, CA). Assays were performed in triplicate. Peptide p-nitroaniline substrate concentrations were determined by absorbance at 342 nm by using an absorption coefficient of 8,266 m–1 cm–1, and product concentrations were calculated by using an absorption coefficient of 9,933 m–1 cm–1 at 405 nm (39Lottenberg R. Hall J. Blinder M. Binder E. Jackson C. Biochim. Biophys. Acta. 1983; 742: 539-557Crossref PubMed Scopus (114) Google Scholar). Active site concentrations ν0=kcat[E]0(1+β[I]0αKi)(1+[I]0αKi)[S]0Km(1+[I]0Ki)(1+[I]0αKi)+[S]0(Eq. 1) for preparations of fXIa were determined by active site titration with human antithrombin in an S2366 cleavage assay. fIX Activation by fXIa—fIX (25–2000 nm) in TBSA containing 5 mm CaCl2 was activated by the addition of fXIa (0.4 nm active sites, 0.2 nm protein). At various time points (0–240 min), 50-μl aliquots were removed and supplemented with aprotinin (final concentration 15 μm) to inhibit fXIa. The steady-state kinetics of hydrolysis of S299 (1 mm) by quenched samples was studied in TBSA containing 5 mm CaCl2 and 33% ethylene glycol. Changes in absorbance at 405 nm were measured on a SpectraMax 340 plate reader. Duplicate assays were run for each fIX concentration. Generation of fIXaβ as a function of time was determined by interpolation from the linear dependence of the initial rate of S299 hydrolysis on known concentrations of fIXaβ. Initial steady-state rates of fIXaβ formation were determined from slopes of plots documenting the linear appearance of fIXaβ with time. Control experiments established that aprotinin completely inhibited fXIa activity without measurable effect on the detection of fIXaβ activity. fIX (50–3000 nm) activation by fXIa was also studied in the presence of aprotinin or fIXai. Aliquots (50 μl) withdrawn at various time points (20–180 s) after the initiation of activation were quenched by the addition of aprotinin (final concentration 15 μm), and fIXaβ generation was determined as described above. All assays were performed in triplicate. Data Analysis—The apparent steady-state kinetic parameters Km and kcat for fIX activation were obtained both by non-linear least-squares fitting of full progress curves for fIX activation at substrate concentrations ranging from 25 to 1000 nm fIX (0.3–11 × Km), taking into account competitive product inhibition by fIXaβ formed during the time courses (40Duggleby R. Morrison J. Biochim. Biophys. Acta. 1977; 481: 297-312Crossref PubMed Scopus (81) Google Scholar), and by the initial rate dependence of fIXaβ formation as a function of fIX concentration. Km and kcat for hydrolysis of S2366 by fXIa were obtained by initial rate analysis of p-nitroaniline generation as a function of S2366 concentration. Ki values for binding of pAB and aprotinin to fXIa were obtained by fitting the substrate and inhibitor dependences of the initial rates of S2366 hydrolysis both by the general hyperbolic mixed-type inhibition model (Scheme 1) and by the classical competitive inhibition model in which no ESI complex is formed (41Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-state Enzyme Systems. John Wiley & Sons, Inc., New York1993: 178-192Google Scholar). The hyperbolic mixed-type inhibition model describes the conversion of S2366 (S) to product (P) by fXIa (E) in the presence of fIX as an inhibitor (I). fIX binds to both free fXIa (E) and fXIa in complex with S2366 (ES) through an exosite interaction. Ks (≈Km) and Ki are dissociation constants for binding of S and I to E, respectively, and kcat is the rate constant for turnover of S in the ES or ESI complex. α and β are the factors by which Ks and kcat change, respectively, when I (fIX) is bound to E. Hyperbolic mixed-type inhibition (Scheme 1) is defined by Equation 1. Equation 1 was used to analyze hydrolysis of S2366 (S) by fXIa (E) in the presence of fIX or fIXai (I), and was compared to the non-competitive (β = 0) model to analyze the cleavage of fIX (S) by fXIa (E) in the presence of aprotinin (I). Binding of fIX or fIXai to fXIa and fXIa·S2366 complex was analyzed by the hyperbolic mixed-type inhibition model and by a two-step interaction model proposed by Boskovic and Krishnaswamy (33Boskovic D. Krishnaswamy S. J. Biol. Chem. 2000; 275: 38561-38570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), shown in Scheme 2. In this model, a macromolecular ligand (S2) interacts at the exosite during the first step, and after a conformational rearrangement governed ν0,P1=kcat[E]0(1+β[S2]0αKS2)(1+[S2]0αKS2)[S1]0kS1(1+[S2]0KS2+[S2]0K*KS2)(1+[S2]0αKS2)+[S1]0(Eq. 2) ν0,P2=kcat[E]0(1+K*([S1]0αKS1))[S2]0K*KS2(1+[S1]0KS1)(1+K*(1+[S1]0αKS1))+[S2]0(Eq. 3) by K*, binds to the active site during the second step. Binding of the macromolecular ligand to the active site is in competition with binding of a small ligand (S1) that reacts with the active site but not with the exosite. Rate equations were derived for this model for turnover of S2366 (S1) proposed to bind only at the active site of fXIa (E) with the dissociation constant KS1 and catalytic constant kcat, and for turnover of fIX (S2) proposed to dock initially at the exosite governed by KS2, and in a second step governed by equilibrium constant K*, with the active site, forming product with the catalytic constant kcat*. The equations were derived without the previous assumption that α = β = 1 (33Boskovic D. Krishnaswamy S. J. Biol. Chem. 2000; 275: 38561-38570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The initial rate of hydrolysis of chromogenic substrate in Michaelis-Menten form is given by Equation 2 and the initial rate of cleavage of fIX is given by Equation 3. Least squares fitting was performed with Scientist software (MicroMath Scientific Software, Salt Lake City, UT), and reported estimates of error represent ± 2 S.D. Inhibition of fXIa-catalyzed Hydrolysis of S2366 by pAB and Aprotinin—fXIa is a homodimer with two active sites (15Bouma B. Griffin J. J. Biol. Chem. 1977; 252: 6432-6437Abstract Full Text PDF PubMed Google Scholar, 16McMullen B. Fujikawa K. Davie E. Biochemistry. 1991; 30: 2056-2060Crossref PubMed Scopus (122) Google Scholar). For all analyses, it is assumed that the active sites function independently of each other, and all protease concentrations given are active site concentrations. The small molecule inhibitor pAB binds reversibly to the S1 substrate-binding subsite of arginine-specific serine proteases (42Bode W. Schwager P. J. Mol. Biol. 1975; 98: 693-717Crossref PubMed Scopus (411) Google Scholar, 43Evans S. Olson S. Shore J. J. Biol. Chem. 1982; 257: 3014-3017Abstract Full Text PDF PubMed Google Scholar) and is an effective active site inhibitor of factor XIa (44Schmidt A. Ogawa T. Gailani D. Bajaj S. J. Biol. Chem. 2004; 279: 29485-29492Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Given its mechanism of action, pAB is expected to be a classical competitive inhibitor of substrate binding to the fXIa active site. The tripeptide substrate S2366 interacts predominantly with the S1–S3 substrate-binding subsites of the active site and was cleaved by fXIa with Km 233 ± 78 μm and kcat 117 ± 10 s–1 (Table I). As expected, pAB was a purely competitive inhibitor of S2366 cleavage with Ki 28 ± 2 μm (Fig. 1, A and B, and Table I). The Kunitz-type inhibitor aprotinin interacts with the fXIa active site and other sites on the catalytic domain but is not expected to interact appreciably with the fXIa heavy chain (45Navaneetham D. Jin L. Babine R. Abdel-Meguid S. Walsh P. Blood. 2003; 102 (Abstr. 435)Google Scholar). Aprotinin behaved as a competitive inhibitor of fXIa cleavage of S2366 with Ki 1.13 ± 0.07 μm (Fig. 1, C and D, and Table I). The data indicate that S2366, pAB, and aprotinin bind to the fXIa active site in a mutually exclusive manner.Table IKinetics of inhibition of fXIa activity by active site inhibitors and by fIX/fIXaiSubstrateInhibitorInhibition typeKmkcatαβKiK*μms-1μmS2366None233 ± 78117 ± 10S2366pABCompetitive233 ± 78117 ± 1028 ± 2S2366aprotininCompetitive233 ± 78117 ± 101.13 ± 0.07S2366fIXMixed hyperbolic233 ± 78117 ± 102.7 ± 0.40.5 ± 0.10.22 ± 0.05S2366fIXTwo step conformational233 ± 78117 ± 102.7 ± 0.40.5 ± 0.1≥5S2366fIXaiMixed hyperbolic233 ± 78117 ± 102.5 ± 0.20.9 ± 0.10.11 ± 0.02S2366fIXaiTwo step conformational233 ± 78117 ± 102.5 ± 0.20.9 ± 0.1≥5fIXNone0.09 ± 0.040.49 ± 0.05fIXAprotininNon-competitive0.09 ± 0.040.49 ± 0.056.3 ± 4.100.89 ± 0.52fIXfIXaiCompetitive0.09 ± 0.040.49 ± 0.050.33 ± 0.05 Open table in a new tab Inhibition of fXIa-catalyzed Hydrolysis of S2366 by fIX and fIXai—In surface plasmon resonance studies, fIX and fIXaβ bind to fXIa with similar affinity (30Aktimur A. Gabriel M. Gailani D. Toomey J. J. Biol. Chem. 2003; 278: 7981-7987Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The effect of fIX and active site-blocked fIXaβ (fIXai) on fXIa hydrolysis of S2366 was investigated. During reactions with fIX, it is expected that some fIXaβ will be generated. Preliminary experiments showed that 2 μm fIXaβ does not hydrolyze S2366 appreciably. If fIX interacts with fXIa exclusively at the active site, it would be expected to behave as a competitive inhibitor of fXIa hydrolysis of S2366. In this case, fIXai interaction at the active site may be weak because of the absence of the activation peptide. On the other hand, if exosite interactions are involved in fIX and fIXai binding to fXIa, mixed-type inhibition of S2366 cleavage would be expected for both molecules. fIX and fIXai inhibited fXIa cleavage of S2366 in a concentration-dependent manner (Fig. 2), and substantial residual fXIa activity remained at saturating fIX or fIXai concentrations. The data were fit well by the hyperbolic mixed-type inhibition model (Scheme 1). Binding of fIX resulted in a 2.7-fold increase in Km and a 50% reduction in kcat for fXIa cleavage of S2366. Ki for binding of fIX to free and S2366-bound fXIa was 0.22 ± 0.05 and 0.59 ± 0.09 μm, respectively (Table I). Binding of fIXai caused a similar 2.5-fold increase in Km and a less explicit (14%) decrease in kcat for S2366 cleavage. Ki for binding of fIXai to free fXIa and to the fXIa·S2366 complex was 0.11 ± 0.02 and 0.28 ± 0.02 μm, respectively (Table I). The Ki for binding of fIXai to fXIa was in good agreement with the Ki for product inhibition obtained from progress curves of fIX hydrolysis by fXIa (0.075 ± 0.015 μm, Fig. 3).Fig. 3Kinetic parameters for fIX cleavage by fXIa.A, initial rates of fIX hydrolysis by 0.4 nm fXIa active sites (vo) as a function of fIX concentration ([fIX]o). B, progress curves of fIXa generation ([fIXa]) from fIX at 25 (∆), 50 (▴), 100 (□), 250 (■), 500 (○), and 1000 nm (●). The lines represent the least-squares fits to the data with the parameters listed in Table I. Rates were measured and analyzed as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) The model was expanded to contain a reversible step for exosite binding followed by docking to the active site, governed by an equilibrium constant K* = [ES]/[ES*] in which [ES] is the fXIa·fIX complex engaged only at the exosite, and [ES*] is the complex engaged at the exosite and active site. A similar model has been described by Boskovic and Krishnaswamy (33Boskovic D. Krishnaswamy S. J. Biol. Chem. 2000; 275: 38561-38570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) (Scheme 2). Using the fixed Ki values from the mixed inhibition model allowed" @default.
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• W2043908422 date "2005-06-01" @default.
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• W2043908422 title "Exosite-mediated Substrate Recognition of Factor IX by Factor XIa" @default.
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• W2043908422 doi "https://doi.org/10.1074/jbc.m500894200" @default.
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