Tryptophan 60-D in the B-insertion loop of thrombin modulates the thrombin-antithrombin reaction.
Tryptophan 60-D in the B-insertion loop of thrombin modulates the thrombin-antithrombin reaction.
复制标题
凝血酶 B 插入环中的色氨酸 60-D 调节凝血酶-抗凝血酶反应。
DOI：
10.1021/bi952065y
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Rezaie,AR
中科院分区：
文献类型：
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作者：
Rezaie,AR
最近的一项研究表明，凝血酶des-PPW与抗凝血酶（AT）的反应非常差。本研究表明，凝血酶的Trp to Ala （W60A）突变体与AT的反应速率也低于凝血酶。用慢结合动力学方法研究了其抑制动力学。在肝素存在和不存在的情况下，凝血酶抑制的伪一级速率常数（kobs）随AT浓度线性增加，表明在所覆盖的浓度范围内，抑制符合双分子反应E + I E-I。只有凝血酶的二级关联速率常数（kassn）可以估计[6.8±2.7)× 103m -1s-1肝素不存在时和（4.1±1.2）× 106m -1s-1肝素存在时]。对于W60A和des-PPW，抑制的koobsof随AT浓度呈双曲线增加，表明抑制是一个按照E + I E·I E-I的两步过程。在肝素不存在时，W60A的动力学常数kinit = 13.6±3.3 μM, k2= 0.007±0.001 s-1；在肝素存在时，kinit = 13.6±3.1 nM, k2= 0.008±0.002 s-1。AT对des-PPW的抑制作用非常缓慢[kassn=（2.9±0.7）× 101M-1s-1]，而肝素对des-PPW的抑制作用加快了约2万倍，并在kinit = 10.4±2.3 nM, k2= 0.006±0.001 s-1时证明了des-PPW的两步反应机制。与凝血酶相比，活性at结合的五糖对des-PPW的抑制作用增强了约15倍。这些结果表明：(1)与凝血酶相反，肝素诱导的AT构象变化是抑制des-PPW的最佳条件；(1)Trp60是正常凝血酶- AT反应所必需的。在这些结果的基础上，提出了一个改进的凝血酶- AT相互作用模型。
In a recent study it was demonstrated that thrombin des-PPW reacts with antithrombin (AT) very poorly. In this study it is shown that a Trp to Ala (W60A) mutant of thrombin also reacts with AT at a lower rate than thrombin. The inhibition kinetics were studied by the slow-binding kinetic approach. In both the presence and absence of heparin, the pseudo-first-order rate constant of thrombin inhibition (kobs) increased linearly with AT concentration, indicating that inhibition, in the concentration range covered, conforms to a bimolecular reaction E + I E-I. Only the second-order association rate constant (kassn) for thrombin can be estimated [6.8 ± 2.7) × 103M-1s-1in the absence of heparin and (4.1 ± 1.2) × 106M-1s-1in the presence of heparin]. With W60A and des-PPW, thekobsof inhibition increased hyperbolically as a function of AT concentration, indicating that the inhibition is a two-step process according to E + I E·I E-I. The kinetic constants for W60A were estimated to beKinit= 13.6 ± 3.3 μM andk2= 0.007 ± 0.001 s-1in the absence of heparin andKinit= 13.6 ± 3.1 nM andk2= 0.008 ± 0.002 s-1in the presence of heparin. AT inhibited des-PPW very slowly [kassn= (2.9 ± 0.7) × 101M-1s-1], but heparin accelerated the reaction ≈20 000-fold and made it possible to demonstrate a two-step reaction mechanism for des-PPW withKinit= 10.4 ± 2.3 nM andk2= 0.006 ± 0.001 s-1. In contrast to thrombin, an active AT-binding pentasaccharide enhanced the inhibition of des-PPW ≈15-fold. These results indicate that (1) in contrast to thrombin, the heparin-induced conformational change in AT is required for optimal inhibition of des-PPW and (1) Trp60 is essential for normal thrombin−AT reaction. On the basis of these results, a modified model for thrombin−AT interaction is proposed.