Krishnaswamy S, Field K A, Edgington T S, Morrissey J H, Mann K G
Department of Biochemistry, University of Vermont, Burlington 05405.
J Biol Chem. 1992 Dec 25;267(36):26110-20.
Coagulation factor X is activated by the extrinsic Xase complex composed of factor VIIa associated with the integral membrane protein tissue factor. The kinetics of human factor X activation was studied following reconstitution of this reaction system using purified human proteins and synthetic phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS) or phosphatidylcholine alone (PC). Factor X activation was evaluated by discontinuous measurements of the amidolytic activity of the product, factor Xa, or continuously monitored using the fluorescent serine protease inhibitor 4-aminobenzamidine. The results of both techniques were verified by direct physical measurements of zymogen activation using SDS-polyacrylamide gel electrophoresis. The rate of factor X activation with PC vesicles was less than 5% of that observed with PCPS vesicles. Since factor X does not bind to vesicles containing only PC, these data suggested an important role for the substrate-membrane interaction in the catalytic cycle. The importance of the substrate-membrane interaction in the activation process was investigated by using membrane-binding proteins to compete with the substrate for combining sites on PCPS vesicles. Prothrombin fragment 1 was an inhibitor of factor X activation. The dependence of inhibition by fragment 1 on PCPS and factor X was consistent with a significant reduction in initial velocity due to the displacement of factor X from the membrane surface. The inhibition data also suggested that the membrane-bound pool of factor X was the preferred substrate for the human extrinsic Xase complex. The influence of PCPS concentrations on the rate of factor X activation was systematically investigated. Increasing concentrations of PCPS resulted in a modest change in the Km,app and a dramatic change in the Vmax,app for the reaction. The initial velocity data could be globally analyzed according to the preferential utilization of membrane-bound factor X with the intrinsic kinetic constants: Km approximately equal to 1 microM and kcat = 37 s-1 at saturating PCPS. In addition, the equilibrium parameters for the factor X-membrane interaction inferred from these studies were in excellent agreement with the directly determined values. Collectively, the data suggest that the substrate-membrane interaction must precede catalysis for the efficient activation of human factor X by the extrinsic Xase complex.
凝血因子X由与整合膜蛋白组织因子相关的因子VIIa组成的外源性X酶复合物激活。使用纯化的人源蛋白和由磷脂酰胆碱和磷脂酰丝氨酸（PCPS）或仅由磷脂酰胆碱（PC）组成的合成磷脂囊泡重建该反应体系后，研究了人凝血因子X激活的动力学。通过间断测量产物凝血因子Xa的酰胺水解活性来评估凝血因子X的激活，或者使用荧光丝氨酸蛋白酶抑制剂4-氨基苯甲脒进行连续监测。两种技术的结果均通过使用SDS-聚丙烯酰胺凝胶电泳对酶原激活进行直接物理测量来验证。用PC囊泡激活凝血因子X的速率不到用PCPS囊泡观察到的速率的5%。由于凝血因子X不与仅含PC的囊泡结合，这些数据表明底物-膜相互作用在催化循环中起重要作用。通过使用膜结合蛋白与底物竞争PCPS囊泡上的结合位点，研究了底物-膜相互作用在激活过程中的重要性。凝血酶原片段1是凝血因子X激活的抑制剂。片段1的抑制作用对PCPS和凝血因子X的依赖性与由于凝血因子X从膜表面移位导致的初始速度显著降低一致。抑制数据还表明，膜结合的凝血因子X池是人类外源性X酶复合物的首选底物。系统研究了PCPS浓度对凝血因子X激活速率的影响。PCPS浓度的增加导致反应的表观Km有适度变化，而表观Vmax有显著变化。初始速度数据可以根据膜结合凝血因子X的优先利用情况，用内在动力学常数进行全局分析：在饱和PCPS时，Km约等于1 microM，kcat = 37 s-1。此外，从这些研究中推断出的凝血因子X-膜相互作用的平衡参数与直接测定的值非常一致。总体而言，数据表明底物-膜相互作用必须先于催化作用，外源性X酶复合物才能有效激活人凝血因子X。
