Rezaie A R
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.
Biochemistry. 1996 Feb 13;35(6):1918-24. doi: 10.1021/bi952065y.
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-->K assn E-I. Only the second-order association rate constant (kassn) for thrombin can be estimated [6.8 +/- 2.7) x 10(3) M-1 s-1 in the absence of heparin and (4.1 +/- 1.2) x 10(6) M-1 s-1 in the presence of heparin]. With W60A and des-PPW, the kobs of inhibition increased hyperbolically as a function of AT concentration, indicating that the inhibition is a two-step process according to E+I<==>K init E.I-->k2 E-I. The kinetic constants for W60A were estimated to be Kinit = 13.6 +/- 3.3 microM and k2 = 0.007 +/- 0.001 s-1 in the absence of heparin and K init = 13.6 +/- 3.1 nM and k2 = 0.008 +/- 0.002 s-1 in the presence of heparin. AT inhibited des-PPW very slowly [K assn = (2.9 +/- 0.7) x 10(1) M-1 s-1], but heparin accelerated the reaction approximately 20,000-fold and made it possible to demonstrate a two-step reaction mechanism for des-PPW with K init = 10.4 +/- 2.3 nM and k2 = 0.006 +/- 0.001 s-1. In contrast to thrombin, an active AT-binding pentasaccharide enhanced the inhibition of des-PPW approximately 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.
最近的一项研究表明，凝血酶去-PPW与抗凝血酶（AT）的反应非常弱。本研究表明，凝血酶的色氨酸至丙氨酸（W60A）突变体与AT的反应速率也低于凝血酶。通过慢结合动力学方法研究了抑制动力学。在有和没有肝素的情况下，凝血酶抑制的假一级速率常数（kobs）均随AT浓度呈线性增加，表明在所涵盖的浓度范围内，抑制符合双分子反应E+I-->K assn E-I。仅可估算凝血酶的二级缔合速率常数（kassn）[在没有肝素的情况下为(6.8±2.7)×10(3) M-1 s-1，在有肝素的情况下为(4.1±1.2)×10(6) M-1 s-1]。对于W60A和去-PPW，抑制的kobs随AT浓度呈双曲线增加，表明抑制是根据E+I<==>K init E.I-->k2 E-I的两步过程。W60A在没有肝素时的动力学常数估计为Kinit = 13.6±3.3 microM和k2 = 0.007±0.001 s-1，在有肝素时为K init = 13.6±3.1 nM和k2 = 0.008±0.002 s-1。AT对去-PPW的抑制非常缓慢[K assn = (2.9±0.7)×10(1) M-1 s-1]，但肝素使反应加速了约20,000倍，并使得能够证明去-PPW的两步反应机制，K init = 10.4±2.3 nM和k2 = 0.006±0.001 s-1。与凝血酶相反，一种活性AT结合五糖使去-PPW的抑制增强了约15倍。这些结果表明：（1）与凝血酶不同，AT中肝素诱导的构象变化是去-PPW最佳抑制所必需的；（2）色氨酸60对于正常的凝血酶-AT反应至关重要。基于这些结果，提出了一种凝血酶-AT相互作用的修正模型。
