Canonical inhibitor-like interactions explain reactivity of α1-proteinase inhibitor Pittsburgh and antithrombin with proteinases
Canonical inhibitor-like interactions explain reactivity of α1-proteinase inhibitor Pittsburgh and antithrombin with proteinases
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DOI：
10.1074/jbc.m305195200
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发表时间：
2003-09-26
影响因子：
4.8
通讯作者：
Gettins, PGW
中科院分区：
文献类型：
--
作者：
Dementiev, A;Simonovic, M;Gettins, PGW
丝氨酸蛋白酶抑制剂抗凝血酶是一种缓慢的凝血酶抑制剂，需要肝素来增强其反应速率。相比之下，α(1)-蛋白酶抑制剂 (α(1)PI) Pittsburgh（P1 Met --> Arg 天然变体）抑制凝血酶的速度比五糖肝素激活的抗凝血酶快 17 倍。我们在这里展示了游离和 S195A 胰蛋白酶结合的 alpha(1)PI Pittsburgh 的 X 射线结构，其表明反应中心环 (RCL) 在游离丝氨酸蛋白酶抑制剂中具有典型构象，该构象在与 S195A 胰蛋白酶结合后不会改变，并且仅在 P2 和 P2' 之间接触蛋白酶。从与S195A凝血酶结合的肝素辅因子II的结构推断，该RCL构象也适合与凝血酶结合。胰蛋白酶和凝血酶与 α(1)PI Pittsburgh 和抗凝血酶及其 P2 变体的反应率表明，低抗凝血酶-凝血酶反应率是由 P2 处的抗凝血酶 RCL 序列引起的，这意味着，在溶液中，抗凝血酶 RCL 必须处于与此处发现的 α(1)PI Pittsburgh 相似的规范构象，即使在非肝素激活状态下也是如此。这表明丝氨酸蛋白酶抑制剂和蛋白酶之间在米氏复合体中存在普遍的、有限的、类似规范的相互作用。
The serpin antithrombin is a slow thrombin inhibitor that requires heparin to enhance its reaction rate. In contrast, alpha(1)-proteinase inhibitor (alpha(1)PI) Pittsburgh (P1 Met --> Arg natural variant) inhibits thrombin 17 times faster than pentasaccharide heparin-activated antithrombin. We present here x-ray structures of free and S195A trypsin-bound alpha(1)PI Pittsburgh, which show that the reactive center loop (RCL) possesses a canonical conformation in the free serpin that does not change upon binding to S195A trypsin and that contacts the proteinase only between P2 and P2'. By inference from the structure of heparin cofactor II bound to S195A thrombin, this RCL conformation is also appropriate for binding to thrombin. Reaction rates of trypsin and thrombin with alpha(1)PI Pittsburgh and antithrombin and their P2 variants show that the low antithrombin-thrombin reaction rate results from the antithrombin RCL sequence at P2 and implies that, in solution, the antithrombin RCL must be in a similar canonical conformation to that found here for alpha(1)PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between serpins and proteinases in their Michaelis complexes.