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Factor VIIa (VIIa) is an unusual trypsin-type serine proteinase that appears to exist in an equilibrium between minor active and dominant zymogen-like inactive conformational states. The binding of tissue factor to VIIa is assumed to shift the equilibrium into the active state. The proteinase domain of VIIa contains a unique structure: a loop formed by a disulfide bond between Cys 310 and Cys 329 , which is five residues longer than those of other trypsin types. To examine the functional role of the loop region, we prepared two mutants of VIIa. One of the mutants, named VII-11, had five extra corresponding residues 316 -320 of VII deleted. The other mutant, VII-31, had all of the residues in its loop replaced with those of trypsin. Functional analysis of the two mutants showed that VIIa-11 (K d ‫؍‬ 41 nM) and VIIa-31 (K d ‫؍‬ 160 nM) had lower affinities for soluble tissue factor as compared with the wild-type VIIa (K d ‫؍‬ 11 nM). The magnitude of tissue factor-mediated acceleration of amidolytic activities of VIIa-11 (7-fold) and that of VIIa-31 (2-fold) were also smaller than that of wild-type VIIa (30-fold). In the absence of tissue factor, VIIa-31 but not VIIa-11 showed enhanced activity; the catalytic efficiencies of VIIa-31 toward various chromogenic substrates were 2-18-fold greater than those of the wildtype VIIa. Susceptibility of the ␣-amino group of Ile-153 of VIIa-31 to carbamylation was almost the same as that of wild-type VIIa, suggesting that VIIa-31 as well as wildtype VIIa exist predominantly in the zymogen-like state. Therefore, the tested modifications in the loop region had adverse effects on affinity for tissue factor, disturbed the tissue factor-induced conformational transition, and changed the catalytic efficiency of VIIa, but they did not affect the equilibrium between active and zymogen-like conformational states.Factor VIIa (VIIa) 1 is a plasma serine proteinase that is essential for the initiation of extrinsic blood coagulation (1). When VIIa forms a complex with tissue factor (TF) in the presence of Ca 2ϩ and phospholipids, the proteinase activity of VIIa toward its natural substrates, factors IX and X, is enhanced by several orders of magnitude, and the coagulation cascade is triggered (2). In vitro, the formation of the active complex can be evidenced by measuring the esterolytic and amidolytic activities of VIIa (3-6); this activity is also enhanced in the presence of soluble TF (sTF) and Ca 2ϩ (4). Human zymogen VII is a single-chain enzyme precursor with an NH 2 -terminal Gla domain (residues 1-39), followed by two EGF-like domains, EGF 1 (residues 50 -81), and EGF 2 (residues 91-127), and a COOH-terminal serine proteinase domain (residues 153-406). Through the limited proteolysis of the Arg 152 -Ile 153 peptide bond, zymogen VII is converted to a twochain form enzyme, activated VII (VIIa), bridged by a disulfide bond (Cys 135 -Cys 262 ), which is composed of a light chain (residues 1-152) with Gla, EGF 1, and EGF 2 domains, and a heavy chain with a serine proteinase domain (re...

To elucidate the functions of the surface loops of VIIa, we prepared two mutants, VII-30 and VII-39. The VII-30 mutant had all of the residues in the 99 loop replaced with those of trypsin. In the VII-39 mutant, both the 99 and 170 loops were replaced with those of trypsin. The k cat /K m value for hydrolysis of the chromogenic peptidyl substrate S-2288 by VIIa-30 (103 mM ؊1 s ؊1 ) was 3-fold higher than that of wild-type VIIa (30.3 mM ؊1 s ؊1 ) in the presence of soluble tissue factor (sTF). This enhancement was due to a decrease in the K m value but not to an increase in the k cat value. On the other hand, the k cat /K m value for S-2288 hydrolysis by VIIa-39 (17.9 mM ؊1 s ؊1 ) was 18-fold higher than that of wild-type (1.0 mM ؊1 s ؊1 ) in the absence of sTF, and the value was almost the same as that of wild-type measured in the presence of sTF. This enhancement was due to not only a decrease in the K m value but also to an increase in the k cat value. These results were in good agreement with their susceptibilities to a subsite 1-directed serine protease inhibitor. In our previous paper (Soejima, K., Mizuguchi, J., Yuguchi, M., Nakagaki, T., Higashi, S., and Iwanaga, S. Coagulation factor VIIa (VIIa) 1 is a plasma serine protease that is essential for the initiation of extrinsic blood coagulation (1). When tissue factor (TF) is expressed after injury of the vessel wall, it forms a complex with factor VIIa, and blood coagulation can be initiated. Forming a complex with TF markedly enhances the ability of VIIa to activate factors IX and X. In vitro, the formation of the active complex can be evidenced by measuring the esterolytic and amidolytic activities of VIIa (2, 3); these activities are also enhanced in the presence of soluble TF (sTF) and calcium ions (3-5).Human zymogen VII is a single chain enzyme precursor with an NH 2 -terminal Gla domain, followed by two EGF-like domains, EGF 1 and EGF 2, and a COOH-terminal serine protease domain. , which is composed of a light chain (residues 1-152) with Gla, EGF 1, and EGF 2 domains, and a heavy chain with a serine protease domain (residues 153-406) (6). The crystal structures of the molecular complex of the active site occupying VIIa, with and without sTF, as well as the crystal structure of zymogen VII, are known (7-12). The shape of VIIa could be described as a tulip, with the catalytic domain as the flower, the light chain as the stem, and the Gla domain as the bulb. TF winds around the light chain (the stem) and the Gla domain (the bulb) of FVII.Conversely, the catalytic domain of the coagulation proteases such as VIIa, IXa, Xa, and ␣-thrombin have active sites and internal cores that are similar to those of trypsin. Although these protease domains have a highly homologous three-dimensional structure, they display significant differences in specificity and catalytic activity. Furthermore, each protease requires a specific cofactor to express enhanced catalytic activity and physiological function, which differs from digestive serine proteases such as trypsin a...