Kvassman J O, Verhamme I, Shore J D
Henry Ford Health Sciences Center, Division of Biochemical Research, Detroit, Michigan, USA.
Biochemistry. 1998 Nov 3;37(44):15491-502. doi: 10.1021/bi9814787.
Serpin inhibitors are believed to form an acyl enzyme intermediate with their target proteinases which is stabilized through insertion of the enzyme-linked part of the reactive center loop (RCL) as strand 4 in beta-sheet A of the inhibitor. To test critically the role and timing of these steps in the reaction of the plasminogen activator inhibitor PAI-1, we blocked the vacant position 4 in beta-sheet A of this serpin with an octapeptide. The peptide-blocked PAI-1 was a substrate for both tissue-type plasminogen activator (tPA) and trypsin and was hydrolyzed at the scissile bond. The reactivity of the peptide-blocked substrate PAI-1 was compared to that of the unmodified inhibitor by rapid acid quenching as well as photometric techniques. With trypsin as target, the limiting rate constants for enzyme acylation were essentially the same with inhibitor and substrate PAI-1 (21-23 s-1), as were also the associated apparent second-order rate constants (2.8-2.9 microM-1 s-1). With tPA, inhibitor and substrate PAI-1 reacted identically to form a tightly bound Michaelis complex (Kd approximately Km approximately 20 nM). The limiting rate constant for acylation of tPA, however, was 57 times faster with inhibitor PAI-1 (3.3 s-1) than with the substrate form (0.059 s-1), resulting in a 5-fold difference in the corresponding second-order rate constants (13 vs 2.5 microM-1 s-1). We attribute the ability of tPA to discriminate between the two PAI-1 forms to exosite bonds that cannot occur with trypsin. The exosite bonds retain specifically the distal part of the PAI-1 RCL in the substrate pocket, which favors a reversal of the acylation step. Acylation of tPA becomes effective only by separating the products of the acylation step. With substrate PAI-1, this depends on passive displacement of bonds, whereas with inhibitor PAI-1, separation is accomplished by loop insertion that pulls tPA from its docking site on PAI-1, resulting in faster acylation than with substrate PAI-1.
丝氨酸蛋白酶抑制剂（Serpin）被认为与其靶蛋白酶形成一种酰基酶中间体，该中间体通过反应中心环（RCL）的酶联部分作为β-折叠A的第4链插入而得以稳定。为了严格测试这些步骤在纤溶酶原激活物抑制剂PAI-1反应中的作用和时机，我们用一个八肽封闭了这种丝氨酸蛋白酶抑制剂β-折叠A中的空缺第4位。肽封闭的PAI-1既是组织型纤溶酶原激活物（tPA）的底物，也是胰蛋白酶的底物，并在可裂解键处被水解。通过快速酸淬灭以及光度技术，将肽封闭的底物PAI-1的反应性与未修饰的抑制剂的反应性进行了比较。以胰蛋白酶为靶标时，酶酰化的极限速率常数在抑制剂和底物PAI-1中基本相同（21 - 23 s⁻¹），相关的表观二级速率常数也相同（2.8 - 2.9 μM⁻¹ s⁻¹）。对于tPA，抑制剂PAI-１和底物PAI-１反应形成紧密结合的米氏复合物（Kd约等于Km约为20 nM）。然而，tPA酰化的极限速率常数在抑制剂PAI-1（3.3 s⁻¹）时比底物形式（0.059 s⁻¹）快57倍，导致相应的二级速率常数相差5倍（13对2.5 μM⁻¹ s⁻¹）。我们将tPA区分两种PAI-1形式（底物和抑制剂）的能力归因于胰蛋白酶不会出现的外部位点键。外部位点键特异性地保留了底物口袋中PAI-1 RCL的远端部分，这有利于酰化步骤的逆转。tPA的酰化仅通过分离酰化步骤的产物才变得有效。对于底物PAI-1，这取决于键的被动置换，而对于抑制剂PAI-1，分离是通过环插入来完成的，环插入将tPA从其在PAI-1上的对接位点拉开，导致酰化比底物PAI-1更快。
