Olson S T, Stephens A W, Hirs C H, Bock P E, Björk I
Center for Molecular Biology of Oral Diseases, University of Illinois-Chicago, Chicago 60612, USA.
J Biol Chem. 1995 Apr 28;270(17):9717-24. doi: 10.1074/jbc.270.17.9717.
To elucidate the role of the P1' residue of the serpin, antithrombin (AT), in proteinase inhibition, the source of the functional defect in a natural Ser-394-->Leu variant, AT-Denver, was investigated. AT-Denver inhibited thrombin, Factor IXa, plasmin, and Factor Xa with second order rate constants that were 430-, 120-, 40-, and 7-fold slower, respectively, than those of native AT, consistent with an altered specificity of the variant inhibitor for its target proteinases. AT-Denver inhibited thrombin and Factor Xa with nearly equimolar stoichiometries and formed SDS-stable complexes with these proteinases, indicating that the diminished inhibitor activity was not due to an enhanced turnover of the inhibitor as a substrate. Binding and kinetic studies showed that heparin binding to AT-Denver as well as heparin accelerations of AT-Denver-proteinase reactions were normal, consistent with the P1' mutation not affecting the heparin activation mechanism. Resolution of the two-step reaction of AT-Denver with thrombin revealed that the majority of the defective function was localized in the second reaction step and resulted from a 190-fold decreased rate constant for conversion of a noncovalent proteinase-inhibitor encounter complex to a stable, covalent complex. Little or no effects of the mutation on the binding constant for encounter complex formation or on the rate constant for stable complex dissociation were evident. These results support a role for the P1' residue of antithrombin in transition-state stabilization of a substrate-like attack of the proteinase on the inhibitor-reactive bond following the formation of a proteinase-inhibitor encounter complex but prior to the conformational change leading to the trapping of proteinase in a stable, covalent complex. Such a role indicates that the P1' residue does not contribute to thermodynamic stabilization of AT-proteinase complexes and instead favors a kinetic stabilization of these complexes by a suicide substrate reaction mechanism.
为阐明丝氨酸蛋白酶抑制剂抗凝血酶（AT）的P1'残基在蛋白酶抑制中的作用，对天然Ser-394→Leu变体AT-丹佛功能缺陷的来源进行了研究。AT-丹佛抑制凝血酶、因子IXa、纤溶酶和因子Xa的二级速率常数分别比天然AT慢430倍、120倍、40倍和7倍，这与变体抑制剂对其靶蛋白酶的特异性改变一致。AT-丹佛以几乎等摩尔的化学计量比抑制凝血酶和因子Xa，并与这些蛋白酶形成SDS稳定的复合物，表明抑制剂活性降低并非由于抑制剂作为底物的周转增强。结合和动力学研究表明，肝素与AT-丹佛的结合以及肝素对AT-丹佛-蛋白酶反应的加速作用正常，这与P1'突变不影响肝素激活机制一致。对AT-丹佛与凝血酶两步反应的解析表明，大部分功能缺陷位于第二步反应，是由于非共价蛋白酶-抑制剂相遇复合物转化为稳定共价复合物的速率常数降低了190倍所致。该突变对相遇复合物形成的结合常数或稳定复合物解离的速率常数几乎没有影响。这些结果支持抗凝血酶的P1'残基在蛋白酶-抑制剂相遇复合物形成后、构象变化导致蛋白酶被困在稳定共价复合物之前，对蛋白酶对抑制剂反应性键的底物样攻击的过渡态稳定中发挥作用。这种作用表明，P1'残基对AT-蛋白酶复合物的热力学稳定没有贡献，而是通过自杀底物反应机制有利于这些复合物的动力学稳定。
