The activation of prothrombin by the prothrombinase complex. The contribution of the substrate-membrane interaction to catalysis.

摘要: The conversion of prothrombin to thrombin requires the cleavage two peptide bonds and is catalyzed by prothrombinase complex composed factors Xa Va assembled on a membrane surface. Presteady-state kinetic studies effects membranes proteolytic reaction were undertaken using model phosphatidylcholine phosphatidylserine (PCPS). concentration PCPS was varied alter free phospholipid available for substrate binding without influencing membrane-assembled prothrombinase. In fluorescence stopped-flow measurements, increasing concentrations resulted in an increase rate product formation. Assessment bond sodium dodecyl sulfate-polyacrylamide gel electrophoresis following rapid chemical quench 125I-prothrombin revealed that activation proceeded through ordered at Arg323-Ile324 followed Art274-Thr275 all PCPS. Increasing large with much smaller effect subsequent Arg274-Thr275, thereby leading extent accumulation intermediate, meizothrombin. Fluorescence measurements also conducted prethrombin 2 plus fragment 1.2 or meizothrombin as substrates assess influence individual reactions. increased approximately 60-fold PCPS, whereas Arg274-Thr275 factor 5. These differential reactions adequately explain changes during activation. Proteolytic removal 1 domain substrates, 2-fragment 1.2, had no saturating but completely eliminated membrane-dependent enhancement Arg323-Ile324. Thus, essential first Arg323-Ile324, which leads contrast, substrate-membrane interaction mediated has detectable second required thrombin.