Boskovic Danilo S, Troxler Thomas, Krishnaswamy Sriram
Joseph Stokes Research Institute, Children's Hospital of Philadelphia, 310A Abramson, 3615 Civic Center Boulevard, Philadelphia, PA 19104, USA.
J Biol Chem. 2004 May 14;279(20):20786-93. doi: 10.1074/jbc.M400469200. Epub 2004 Feb 26.
The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green(488) at the active site of prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with prothrombinase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of prothrombin activation and product release.
凝血酶原向凝血酶的转化由凝血酶原酶催化，凝血酶原酶是一种酶复合物，由丝氨酸蛋白酶因子Xa和辅因子蛋白因子Va组成，组装在膜上。动力学研究表明，与扩展的大分子识别位点（外位点）而非凝血酶原酶的活性位点相互作用是决定底物或产物结合亲和力的主要因素。我们现在通过对底物衍生物和产物与凝血酶原酶相互作用的物理研究，对这些观点进行了独立于模型的评估。使用用荧光肽基氯甲基酮修饰的Xa组装酶复合物，以不可逆地封闭活性位点。通过凝血酶原酶活性位点上Oregon Green(488)荧光的凝血酶原2依赖性扰动推断结合。通过连接到Xa活性位点的2,6-丹磺酰基与连接到凝血酶或中凝血酶去片段1活性位点的曙红之间的荧光共振能量转移，也明确证实了不依赖活性位点的结合。从这些测量中获得的可比探针间距离表明，底物和产物与酶的相互作用方式相同。竞争实验确定了一系列底物或产物衍生物以相互排斥的方式结合到凝血酶原酶上的能力。凝血酶原、凝血酶原2、中凝血酶去片段1和凝血酶与凝血酶原酶的不依赖活性位点结合的平衡解离常数与使用活性酶的动力学研究推断的亲和力相当。我们的研究结果直接表明，结合亲和力主要由底物、两种可能的中间体或产物与凝血酶原酶的外位点介导的相互作用决定。单一类型的外位点结合相互作用显然通过凝血酶原激活和产物释放的两个裂解反应所需的逐步反应驱动亲和力和结合特异性。
