科研之友
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摘要
Heparin activates the serpin, antithrombin, to inhibit its target blood-clotting proteases by generating new protease interaction exosites. To resolve the effects of these exosites on the initial Michaelis docking step and the subsequent acylation and conformational change steps of antithrombin-protease reactions, we compared the reactions of catalytically inactive S195A and active proteases with site-specific fluorophore-labeled antithrombins that allow monitoring of these reaction steps. Heparin bound to N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-3-oxa-1,3diazol- 4-yl)ethylenediamine (NBD)-fluorophore-labeled antithrombins and accelerated the reactions of the labeled inhibitor with thrombin and factor Xa similar to wild type. Equilibrium binding of NBD- labeled antithrombins to S195A proteases showed that exosites generated by conformationally activating antithrombin with a heparin pentasaccharide enhanced the affinity of the serpin for S195A factor Xa minimally 100-fold. Moreover, additional bridging exosites provided by a hexadecasaccharide heparin activator enhanced antithrombin affinity for both S195A factor Xa and thrombin at least 1000-fold. Rapid kinetic studies showed that these exosite-mediated enhancements in Michaelis complex affinity resulted from increases in k(on) and decreases in k(off) and caused antithrombin-protease reactions to become diffusion-controlled. Competitive binding and kinetic studies with exosite mutant antithrombins showed that Tyr-253 was a critical mediator of exosite interactions with S195A factor Xa; that Glu-255, Glu-237, and Arg-399 made more modest contributions to these interactions; and that exosite interactions reduced koff for the Michaelis complex interaction. Together these results show that exosites generated by heparin activation of antithrombin function both to promote the formation of an initial antithrombin-protease Michaelis complex and to favor the subsequent acylation of this complex.
- 出版日期2007-11-16
