Goodwin C A, Deadman J J, Le Bonniec B F, Elgendy S, Kakkar V V, Scully M F
Thrombosis Research Institute, Emmanuel Kaye Building, London, U.K.
Biochem J. 1996 Apr 1;315 ( Pt 1)(Pt 1):77-83. doi: 10.1042/bj3150077.
The thrombin mutant, des-ETW-thrombin, lacking Glu(146), Thr(147), and Trp(148) within a unique insertion loop located at the extreme end of the primary specificity pocket, has been shown previously to exhibit reduced catalytic activity with respect to macromolecular and synthetic thrombin substrates and reduced or enhanced susceptibility to inhibition. Investigation of the hydrolysis of peptidyl p-nitroanilide substrates by des-ETW-thrombin showed increased activity in the presence of heparin and other sulphated glycosaminoglycans. No effect was observed upon the activity of wild-type thrombin. Heparin was found to decrease the K(m) for cleavage of four thrombin-specific substrates by des-ETW-thrombin by 3-4-fold. Similarly, pentosan polysulphate (PPS) decreased the K(m) with these substrates by 8-10-fold. Heparin also increased the rate of inhibition of des-ETW-thrombin by antithrombin III and D-phenylalanyl-prolyl-arginylchloromethane (PPACK). The inhibition of des-ETW-thrombin by a number of thrombin-specific peptide boronic acids also showed significant reduction in the final K(i) in the presence of heparin, due to reduction in the off-rate. A peptide analogue of a sequence of hirudin which binds thrombin tightly to exosite I (fibrinogen recognition site) potentiated the activity of des-ETW-thrombin against peptide p-nitroanilide substrates in a manner similar to heparin. The K(i) for the inhibition of des-ETW-thrombin by p-aminobenzamidine was decreased by these ligands from 9.7 mM to 7.5 mM, 5.1 mM, and 2.5 mM in the presence of heparin, hirudin peptide and PPS respectively, suggesting the increased catalytic activity is due to enhanced access to the primary specificity pocket. The positive influence of these ligands on des-ETW-thrombin was reversed in the presence of ATP or ADP; the latter has previously been shown to inhibit thrombin activity by blocking initial interaction with fibrinogen at exosite 1. Because the effect of heparin and PPS is similar to that of hirudin peptide, it is proposed that the most likely mechanism is that binding at the heparin-binding site (thrombin exosite 2) facilitates interaction at exosite 1 causing a conformational change which partially corrects the defective ground-state binding of the mutant thrombin. Although no effect was observed upon the activity of wild-type thrombin, our findings do provide further evidence of an allosteric property of thrombin which may control the geometry of, and access to, the primary specificity pocket.
凝血酶突变体des-ETW-凝血酶在位于主要特异性口袋末端的独特插入环内缺少Glu(146)、Thr(147)和Trp(148)，先前已表明其对大分子和合成凝血酶底物的催化活性降低，对抑制的敏感性降低或增强。对des-ETW-凝血酶水解肽基对硝基苯胺底物的研究表明，在肝素和其他硫酸化糖胺聚糖存在下活性增加。野生型凝血酶的活性未观察到影响。发现肝素可使des-ETW-凝血酶切割四种凝血酶特异性底物的K(m)降低3至4倍。同样，戊聚糖多硫酸盐（PPS）可使这些底物的K(m)降低8至10倍。肝素还增加了抗凝血酶III和D-苯丙氨酰-脯氨酰-精氨酰氯甲烷（PPACK）对des-ETW-凝血酶的抑制速率。在肝素存在下，许多凝血酶特异性肽硼酸对des-ETW-凝血酶的抑制作用也显示最终K(i)显著降低，这是由于解离速率降低。紧密结合凝血酶至外位点I（纤维蛋白原识别位点）的水蛭素序列的肽类似物以类似于肝素的方式增强了des-ETW-凝血酶对肽对硝基苯胺底物的活性。在肝素、水蛭素肽和PPS存在下，对氨基苯甲脒抑制des-ETW-凝血酶的K(i)分别从9.7 mM降至7.5 mM、5.1 mM和2.5 mM，表明催化活性增加是由于更容易进入主要特异性口袋。在ATP或ADP存在下，这些配体对des-ETW-凝血酶的积极影响被逆转；后者先前已表明通过阻断在外位点1与纤维蛋白原的初始相互作用来抑制凝血酶活性。由于肝素和PPS的作用与水蛭素肽相似，因此提出最可能的机制是在肝素结合位点（凝血酶外位点2）的结合促进了在外位点1的相互作用，导致构象变化，部分纠正了突变凝血酶有缺陷的基态结合。虽然野生型凝血酶的活性未观察到影响，但我们的发现确实为凝血酶的变构特性提供了进一步证据，该变构特性可能控制主要特异性口袋的几何形状和进入该口袋的途径。
