Factor Xa (FXa) hydrolyzes two peptide bonds in prothrombin having (Glu/Asp)-Gly-Arg-(Thr/Ile) for P 3 -P 2 -P 1 -P 1 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 P 3 , P 2 , P 1 , P 2 , and P 3 and determined the k cat /K m values of cleavage. Contrary to expectation, glycine was not the "best" P 2 residue; peptide with phenylalanine was cleaved slightly faster. In fact, FXa had surprisingly limited preferences, barely more pronounced than trypsin; in P 2 , the ratio of the k cat /K m values for the most favorable side chain over the least was 289 (12 with trypsin), but in P 1 , 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 P 2 and P 1 . 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) 1 is the midway protease of the blood clotting waterfall (1). FXa belongs to clan SA of the S1 family of serine peptidases along with thrombin and trypsin (2-5). Without cofactors, activation of prothrombin by FXa is slow; it becomes efficient only when FXa complexes factor Va to form prothrombinase (6). Rapid inhibition of FXa by antithrombin also requires heparin as cofactor (7). However, tissue factor pathway inhibitor (TFPI) does not require any cofactor to rapidly neutralize FXa (8). FXa catalyzes a number of other reactions: activation of factor VII in a positive feedback within the tissue factor pathway (9), activation of factors V (10) and VIII (11), cleavage of protease-activated receptor 2 (12), and neutralization of protein S, albeit only in the presence of phospholipid and calcium (13). Thrombin (14) 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 (6, 10, 15). 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 molecul...

Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P 14 to P 9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P 1 and P 1 residues). Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHK, located in native antithrombin on strand 4 of ␤-sheet A, which becomes strand 5 of ␤-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P 14 to P 6 of the RCL are inserted into ␤-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex. Serine protease inhibitors (serpins)1 are mainly composed of three ␤-sheets (A, B, and C) united by nine ␣-helices (A-I); indeed, many are inhibitors that neutralize their target(s) by forming a, stoichiometric, stable complex (1-3). Formation of the stable complex involves the charge stabilizing system of the target protease (4 -8) and a surface loop of the inhibitor called the reactive center loop (RCL). The RCL connects strand 4 of ␤-sheet A to strand 1 of ␤-sheet C; it is exposed to solvent in the inhibitory serpins. By analogy with protease substrates, the 20 amino acids constituting the RCL are numbered P n -. . . -P 1 -PЈ 1 -. . . -PЈ n , where P 1 -PЈ 1 is ultimately cleaved. The mechanism of protease inhibition involves multiple steps that initiate by the formation of a reversible association, converting to a stable complex, ultimately split into regenerated enzyme and RCL-cleaved (consumed) serpin (9 -12). The RCL sustains a variety of conformations (13-16). In antithrombin (AT) that is heparin-activated (17, 18) and other inhibitory serpins such as ␣1-antitrypsin (also called ␣1-proteinase inhibitor; 19 -21) or ␣1-antichymotrypsin (22), the RCL is wholly exposed, whereas in the AT monomer, residue P 14 of the RCL disrupts ␤-sheet A (23-25). In latent AT, an intact but non-inhibitory conformer (25), and in latent type-1 plasminogen activator inhibitor (16), residues P 14 to P 3 of the RCL are completely inserted into ␤-sheet A, constituting an additional, sixth, strand. The same conversion from a five-to six-stranded ␤-sheet A occurs in the inhibitory serpins, following cleavage of the RCL (14, 26).To date, no x-ray analysis of a serpin-protease complex has been reported; thus, its structure rema...

Thrombin is an allosteric protease controlled through exosites flanking the catalytic groove. Binding of a peptide derived from hirudin (Hir 52-65 ) and/or of heparin to these opposing exosites alters catalysis. We have investigated the contribution of subsites S 2 and S 3 to this allosteric transition by comparing the hydrolysis of two sets of fluorescence-quenched substrates having all natural amino acids at positions P 2 and P 3 . Regardless of the amino acids, Hir 52-65 decreased, and heparin increased the k cat /K m value of hydrolysis by thrombin. Several lines of evidence have suggested that Glu 192 participates in this modulation. We have examined the role of Glu 192 by comparing the catalytic activity of thrombin and its E192Q mutant. Mutation substantially diminishes the selectivity of thrombin. The substrate with the "best" P 2 residue was cleaved with a k cat /K m value only 49 times higher than the one having the "least favorable" P 2 residue (versus 636-fold with thrombin). Mutant E192Q also lost the strong preference of thrombin for positively charged P 3 residues and its strong aversion for negatively charged P 3 residues. Furthermore, both Hir 52-65 and heparin increased the k cat /K m value of substrate hydrolysis. We conclude that Glu 192 is critical for the P 2 and P 3 specificities of thrombin and for the allostery mediated through exosite 1.Thrombin (1, 2) is a multifunction serine protease finely tuned through: 1) restrictions fulfilled by the nature of the P 3 to P 3 Ј residues of the substrate, 1 that the S 3 to S 3 Ј subsites of the protease must accommodate, 2) steric hindrance resulting from surface loops, that limit access to the active site, 3) secondary exosites (one apolar and two positively charged) that strengthen the binding of cognate macromolecules (e.g. substrates such as fibrinogen but also inhibitors such as hirudin), and 4) allosteric control conferred by various cofactors (3). Several lines of evidence suggest that Glu 192 is a major player in the specificity of thrombin (4).2 Overall, hydrolysis by thrombin of its "best" substrates is little affected by the E192Q mutation, but new activities emerge that are severely restrained in normal thrombin. The E192Q mutant activates bovine factor X (5) and is efficiently inactivated by the serpin ␣ 1 -antitrypsin (6) and by the bovine pancreatic trypsin inhibitor (BPTI) 3 (7, 8). Glu 192 also seems to participate in the allosteric modulation of thrombin (4, 9). E192Q activates the anticoagulant protein C faster than thrombin; however, in the presence of the cofactor thrombomodulin, this difference disappears (4). Notably, the side chain of Glu 192 changes its conformation upon binding of a ligand to exosite 1 (10 -12).Among the effectors altering thrombin catalysis, molecules as diverse as inorganic ions, tryptamine analogs, polysaccharides, and proteins (or peptides derived from these proteins) have been identified. Sodium ions are allosteric modulators of thrombin, along with other serine proteases having a tyrosine in position...