Boskovic D S, Krishnaswamy S
Joseph Stokes Research Institute, Children's Hospital of Philadelphia Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2000 Dec 8;275(49):38561-70. doi: 10.1074/jbc.M006637200.
The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg(323)-Ile(324), followed by cleavage at Arg(274)-Thr(275). We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIaDeltaF1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIaDeltaF1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to K(m). This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIaDeltaF1. As thrombin is known to bind to an exosite on prothrombinase, initial interactions at an exosite likely play a role in the enzyme-substrate interaction. Occupation of the active site of prothrombinase by a reversible inhibitor does not exclude the binding of mIIaDeltaF1 to the enzyme. Specific recognition of mIIaDeltaF1 is achieved through an initial bimolecular reaction with an enzymic exosite, followed by an active site docking step in an intramolecular reaction prior to bond cleavage. By alternate substrate studies, we have resolved the contributions of the individual binding steps to substrate affinity and catalysis. This pathway for substrate binding is identical to that previously determined with a substrate analog for the first half-reaction of prothrombin activation. We show that differences in the observed kinetic constants for the two cleavage reactions arise entirely from differences in the inferred equilibrium constant for the intramolecular binding step that permits elements surrounding the scissile bond to dock at the active site of prothrombinase. Therefore, substrate specificity is achieved by binding interactions with an enzymic exosite that tethers the protein substrate to prothrombinase and directs cleavage at two spatially distinct scissile bonds.
凝血酶原酶复合物由蛋白酶因子Xa与膜上的因子Va结合组成，通过在精氨酸（323）-异亮氨酸（324）处对凝血酶原进行特异性且有序的蛋白水解，接着在精氨酸（274）-苏氨酸（275）处裂解，从而催化凝血酶的形成。我们使用了凝血酶原片段1缺失型中凝血酶（mIIaDeltaF1）的荧光衍生物作为底物类似物，来评估牛凝血酶原激活后半反应中底物识别的机制。无论底物浓度相对于米氏常数（K(m)）如何，mIIaDeltaF1的裂解均呈现伪一级动力学。这种现象源于凝血酶的竞争性产物抑制，凝血酶与凝血酶原酶的结合亲和力与mIIaDeltaF1完全相同。由于已知凝血酶与凝血酶原酶上的一个外位点结合，外位点处的初始相互作用可能在酶-底物相互作用中发挥作用。用可逆抑制剂占据凝血酶原酶的活性位点并不排除mIIaDeltaF1与该酶的结合。mIIaDeltaF1的特异性识别是通过与酶外位点的初始双分子反应实现的，随后在分子内反应中进行活性位点对接步骤，然后再进行键裂解。通过替代底物研究，我们解析了各个结合步骤对底物亲和力和催化作用的贡献。这种底物结合途径与先前用凝血酶原激活前半反应的底物类似物所确定的途径相同。我们表明，两个裂解反应观察到的动力学常数差异完全源于分子内结合步骤推断的平衡常数差异，该步骤允许裂解键周围的元件在凝血酶原酶的活性位点对接。因此，底物特异性是通过与酶外位点的结合相互作用实现的，这种相互作用将蛋白质底物束缚于凝血酶原酶，并指导在两个空间上不同的裂解键处进行裂解。
