Rezaie A R, Esmon C T
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City 73104, USA.
Eur J Biochem. 1996 Dec 15;242(3):477-84. doi: 10.1111/j.1432-1033.1996.477rr.x.
Residue 192 (chymotrypsin numbering system) in thrombin, activated protein C, and factor Xa contributes to the specificity of these enzymes toward their substrates and inhibitors. A Glu192-->Gln mutation in both thrombin and activated protein C yielded enzymes that reacted better with some, but not all, of their natural substrates and inhibitors. To determine whether the specificity change is due to productive interactions of Gln192 with substrates and inhibitors or elimination of repulsive electrostatic interactions, we prepared forms of thrombin, des-(1-45)-factor Xa and activated des-(1-45)-protein C with Glu, Gln, or Met at position 192 and compared their activities toward inhibitors and substrates. All mutants had nearly normal amidolytic activity. The Glu192-->Gln and Glu 192-->Met mutations of thrombin and activated des-(1-45)-protein C increased the second-order rate constant (k2) of inhibition by alpha 1-antitrypsin about 700-fold and 170-fold for thrombin, and 185-fold and 150-fold for activated des-(1-45)-protein C, respectively. [E192]faxtor Xa, but not [M192]factor Xa, was resistant to inhibition by alpha 1-antitrypsin. Glu-->Gln or Glu-->Met mutants of both thrombin and activated des-(1-45)-protein C were effectively inhibited by tissue factor pathway inhibitor (K1 < 200 nM) and, except for [M192]thrombin, by bovine pancreatic trypsin inhibitor (K1 < 60 nM). With respect to substrate cleavage, Glu192-->Gln and Glu192-->Met mutations of activated des-(1-45)-protein C both inactivated factor Va 2-3-fold faster than activated des-(1-45)-protein C. Thrombin and [M192]thrombin activated protein C at similar slow rates compared to rapid activation by [Q192]thrombin. The Gln192-->Met mutants of des-(1-45)-factor Xa activated prethrombin 1.8-11-fold slower than wild-type enzyme. With thrombomodulin or factor Va present, these differences in protein C and prethrombin 1 activation rates were decreased to about 2-fold. We conclude that residue 192 contribution to enzyme specificity is achieved by both productive and repulsive interactions and that the magnitude and nature of the participation varies among enzymes, substrates and inhibitors.
凝血酶、活化蛋白C和因子Xa中的192位残基（胰凝乳蛋白酶编号系统）决定了这些酶对其底物和抑制剂的特异性。凝血酶和活化蛋白C中第192位的谷氨酸突变为谷氨酰胺后，所产生的酶与部分而非全部天然底物和抑制剂的反应性增强。为确定特异性变化是由于谷氨酰胺192与底物和抑制剂之间的有效相互作用，还是由于排斥性静电相互作用的消除，我们制备了192位分别为谷氨酸、谷氨酰胺或甲硫氨酸的凝血酶、去（1 - 45）-因子Xa和活化去（1 - 45）-蛋白C，并比较了它们对抑制剂和底物的活性。所有突变体的酰胺水解活性几乎正常。凝血酶和活化去（1 - 45）-蛋白C的谷氨酸192突变为谷氨酰胺和谷氨酸192突变为甲硫氨酸后，α1 -抗胰蛋白酶对它们的抑制二级速率常数（k2）分别增加了约700倍和170倍（凝血酶），以及185倍和150倍（活化去（1 - 45）-蛋白C）。[E192]因子Xa对α1 -抗胰蛋白酶的抑制有抗性，而[M192]因子Xa则无。凝血酶和活化去（1 - 45）-蛋白C的谷氨酸突变为谷氨酰胺或谷氨酸突变为甲硫氨酸的突变体均能被组织因子途径抑制剂有效抑制（K1 < 200 nM），除了[M192]凝血酶外，也能被牛胰蛋白酶抑制剂抑制（K1 < 60 nM）。在底物裂解方面，活化去（1 - 45）-蛋白C的谷氨酸192突变为谷氨酰胺和谷氨酸192突变为甲硫氨酸的突变体使因子Va失活的速度比活化去（1 - 45）-蛋白C快2 - 3倍。与[Q192]凝血酶的快速激活相比，凝血酶和[M192]凝血酶激活蛋白C的速度较慢且相似。去（1 - 45）-因子Xa的谷氨酰胺192突变为甲硫氨酸的突变体激活凝血酶原的速度比野生型酶慢1.8 - 11倍。当存在血栓调节蛋白或因子Va时，蛋白C和凝血酶原1激活速率的这些差异降至约2倍。我们得出结论，192位残基对酶特异性的贡献是通过有效相互作用和排斥相互作用共同实现的，并且参与的程度和性质在不同的酶、底物和抑制剂之间有所不同。
