Soejima K, Mizuguchi J, Yuguchi M, Nakagaki T, Higashi S, Iwanaga S
First Research Department, The Chemo-Sero-Therapeutic Research Institute, Kumamoto 869-1298, the Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan.
J Biol Chem. 2001 May 18;276(20):17229-35. doi: 10.1074/jbc.M009206200. Epub 2001 Feb 2.
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 Cys310 and Cys329, 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 (Kd = 41 nm) and VIIa-31 (Kd = 160 nm) had lower affinities for soluble tissue factor as compared with the wild-type VIIa (Kd = 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 wild-type VIIa. Susceptibility of the alpha-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 wild-type 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.
凝血因子VIIa（VIIa）是一种特殊的胰蛋白酶型丝氨酸蛋白酶，似乎在次要活性构象状态和主要的类酶原无活性构象状态之间处于平衡。组织因子与VIIa的结合被认为会使平衡向活性状态转变。VIIa的蛋白酶结构域具有独特的结构：由半胱氨酸310和半胱氨酸329之间的二硫键形成的一个环，比其他胰蛋白酶类型的环长五个残基。为了研究环区域的功能作用，我们制备了两种VIIa突变体。其中一个突变体名为VII-11，缺失了VII的316 - 320位五个额外的相应残基。另一个突变体VII-31，其环中的所有残基都被胰蛋白酶的残基取代。对这两种突变体的功能分析表明，与野生型VIIa（解离常数Kd = 11 nm）相比，VIIa-11（Kd = 41 nm）和VIIa-31（Kd = 160 nm）对可溶性组织因子的亲和力较低。VIIa-11（7倍）和VIIa-31（2倍）的组织因子介导的酰胺水解活性加速幅度也小于野生型VIIa（30倍）。在没有组织因子的情况下，VIIa-31而不是VIIa-11表现出增强的活性；VIIa-31对各种显色底物的催化效率比野生型VIIa高2 - 18倍。VIIa-31的异亮氨酸153的α-氨基对氨甲酰化的敏感性与野生型VIIa几乎相同，表明VIIa-31以及野生型VIIa主要以类酶原状态存在。因此，环区域中经过测试的修饰对组织因子的亲和力有不利影响，扰乱了组织因子诱导的构象转变，并改变了VIIa的催化效率，但它们不影响活性构象状态和类酶原构象状态之间的平衡。
