Fredenburgh J C, Stafford A R, Weitz J I
Hamilton Civic Hospitals Research Centre, and McMaster University, Hamilton, Ontario, Canada L8V 1C3.
J Biol Chem. 1997 Oct 10;272(41):25493-9. doi: 10.1074/jbc.272.41.25493.
Investigations to date have demonstrated that ligand binding to exosites 1 or 2 on thrombin produces conformational changes at the active site. In this study, we directly compared the effect of ligand binding to exosites 1 and 2 on the structure and function of the active site of thrombin and investigated functional linkage between the two exosites. Binding studies were performed in solution with fluorescein-Phe-Pro-Arg-CH2Cl (FPR)-thrombin. Hirudin-(54-65) and sF2, a synthetic peptide corresponding to residues 63-116 of prothrombin fragment 2, were used as ligands for exosites 1 and 2 of thrombin, respectively. The two ligands produce diametric changes in the fluorescence of fluorescein-FPR-thrombin and also have opposing effects on the rate of thrombin hydrolysis of a number of chromogenic substrates. These results indicate that sF2 and hirudin-(54-65) differentially affect the conformation of the active site. Experiments then were performed to investigate whether both ligands can bind to thrombin simultaneously. When thrombin-bound fluorescein-sF2 is titrated with hirudin-(54-65), complete displacement of fluorescein-sF2 is observed. Likewise, when thrombin-bound fluorescein-hirudin-(54-65) is titrated with sF2, complete displacement occurs. Additional support for reciprocal binding was obtained in fluorescence experiments where both probes were labeled and in experiments monitoring ligand binding to agarose-immobilized thrombin. This mutually exclusive binding of either ligand can be explained by reciprocal, allosteric modulation of ligand affinity between the two exosites. Thus, not only do the two exosites differentially influence the active site, they also affect the binding properties of the opposing exosite.
迄今为止的研究表明，配体与凝血酶上的外位点1或2结合会在活性位点产生构象变化。在本研究中，我们直接比较了配体与外位点1和2结合对凝血酶活性位点结构和功能的影响，并研究了两个外位点之间的功能联系。在溶液中用荧光素-Phe-Pro-Arg-CH2Cl（FPR）-凝血酶进行结合研究。水蛭素-（54-65）和sF2（一种与凝血酶原片段2的63-116位残基对应的合成肽）分别用作凝血酶外位点1和2的配体。这两种配体使荧光素-FPR-凝血酶的荧光产生完全相反的变化，并且对多种生色底物的凝血酶水解速率也有相反的影响。这些结果表明，sF2和水蛭素-（54-65）对活性位点的构象有不同的影响。然后进行实验以研究两种配体是否能同时与凝血酶结合。当用水蛭素-（54-65）滴定结合了凝血酶的荧光素-sF2时，观察到荧光素-sF2被完全置换。同样，当用sF2滴定结合了凝血酶的荧光素-水蛭素-（54-65）时，也会发生完全置换。在两种探针都被标记的荧光实验以及监测配体与琼脂糖固定化凝血酶结合的实验中，获得了相互结合的额外证据。两种配体的这种相互排斥结合可以通过两个外位点之间配体亲和力的相互变构调节来解释。因此，这两个外位点不仅对活性位点有不同的影响，它们还会影响相对外位点的结合特性。
