Identification of a binding site for quaternary amines in factor Xa.
Identification of a binding site for quaternary amines in factor Xa.
复制标题
鉴定因子 Xa 中季胺的结合位点。
作者：
D. Monnaie;D. Arosio;N. Griffon;T. Rose;A. Rezaie;E. Di Cera
在表征因子XA的Na（+）结合特性的过程中，发现季胺对该酶具有特异性抑制作用，这与之前的观察结果一致。在低毫摩尔范围内与K（i）发生结合，其中三甲基苯基铵（TMPA）显示出最高的特异性。TMPA的结合以竞争性方式抑制底物水解，不抑制对氨基苯甲脒与S1口袋的结合，并与Na（+）结合呈正相关。在凝血酶和组织纤溶酶原激活物（tPA）中也观察到TMPA的抑制作用，但与因子Xa相比程度较低。使用因子Xa的晶体结构的计算机建模表明，TMPA与S2/S3特异性位点结合，其疏水部分与Y99、F174和W215的侧链产生货车范德华相互作用，并且带电胺与E97的羧酸盐静电偶联。因子Xa，凝血酶和tPA的定点突变证实了由对接计算得出的预测，并揭示了残基Y99的主导作用。TMPA与Xa因子的结合通过Y99T置换而显著降低（25倍）。同样，Y99L取代损害TMPA与tPA的结合。另一方面，TMPA的亲和力在具有取代L99Y的凝血酶中增强4倍。Xa因子中季胺结合位点的确定与该凝血酶的选择性抑制剂的合理设计有关。
In the process of characterizing the Na(+)-binding properties of factor Xa, a specific inhibition of this enzyme by quaternary amines was identified, consistent with previous observations. The binding occurs with K(i) in the low millimolar range, with trimethylphenylammonium (TMPA) showing the highest specificity. Binding of TMPA inhibits substrate hydrolysis in a competitive manner, does not inhibit the binding of p-aminobenzamidine to the S1 pocket, and is positively linked to Na(+) binding. Inhibition by TMPA is also seen in thrombin and tissue plasminogen activator (tPA), though to a lesser extent compared to factor Xa. Computer modeling using the crystal structure of factor Xa suggests that TMPA binds to the S2/S3 specificity sites, with its hydrophobic moiety making van der Waals interactions with the side chains of Y99, F174, and W215, and the charged amine coupling electrostatically with the carboxylates of E97. Site-directed mutagenesis of factor Xa, thrombin, and tPA confirms the predictions drawn by docking calculations and reveal a dominant role for residue Y99. Binding of TMPA to factor Xa is drastically (25-fold) reduced by the Y99T replacement. Likewise, the Y99L substitution compromises binding of TMPA to tPA. On the other hand, the affinity of TMPA is enhanced 4-fold in thrombin with the substitution L99Y. The identification of a binding site for quaternary amines in factor Xa has a bearing on the rational design of selective inhibitors of this clotting enzyme.