The activation of prothrombin by the prothrombinase complex. The contribution of the substrate-membrane interaction to catalysis.
The conversion of prothrombin to thrombin requires the cleavage of two peptide bonds and is catalyzed by the prothrombinase complex composed of factors Xa and Va assembled on a membrane surface. Presteady-state kinetic studies of the effects of membranes on the proteolytic reaction were undertaken using model membranes composed of phosphatidylcholine and phosphatidylserine (PCPS). The concentration of PCPS was varied to alter the concentration of free phospholipid available for substrate binding without influencing the concentration of membrane-assembled prothrombinase. In fluorescence stopped-flow measurements, increasing concentrations of PCPS resulted in an increase in the rate of product formation. Assessment of bond cleavage by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following rapid chemical quench using 125I-prothrombin revealed that the activation reaction proceeded through the ordered cleavage at Arg323-Ile324 followed by cleavage at Art274-Thr275 at all concentrations of PCPS. Increasing the PCPS concentration resulted in a large increase in the Arg323-Ile324 cleavage reaction with a much smaller effect on the subsequent cleavage at Arg274-Thr275, thereby leading to an increase in the extent of accumulation of the intermediate, meizothrombin. Fluorescence stopped-flow and rapid chemical quench measurements were also conducted using prethrombin 2 plus fragment 1.2 or meizothrombin as substrates to assess the influence of PCPS on the individual cleavage reactions. The rate of cleavage at Arg323-Ile324 by prothrombinase was increased approximately 60-fold with increasing PCPS, whereas the cleavage at Arg274-Thr275 was increased by a factor of approximately 5. These differential effects of PCPS on the two cleavage reactions adequately explain changes in the extent of accumulation of meizothrombin during prothrombin activation. Proteolytic removal of the membrane binding fragment 1 domain of the substrates, meizothrombin and prethrombin 2-fragment 1.2, had no effect on the cleavage at Arg274-Thr275 at saturating PCPS but completely eliminated the membrane-dependent rate enhancement for cleavage at Arg323-Ile324. Thus, membrane binding by the substrate is essential for the first cleavage reaction at Arg323-Ile324, which leads to the conversion of prothrombin to meizothrombin. In contrast, the substrate-membrane interaction mediated by the fragment 1 domain has no detectable effect on the second cleavage reaction at Arg274-Thr275 which is required for the conversion of meizothrombin to thrombin.
Walker
R K
RK
Department of Medicine, Emory University, Atlanta, Georgia 30322.
Krishnaswamy
S
S
D000818:Animals; D002384:Catalysis; D002417:Cattle; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D015943:Factor Va / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*
D004792:Enzyme Precursors; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D015943:Factor Va; D011516:Prothrombin; D013925:Thromboplastin; D013917:Thrombin; C026812:meizothrombin; D015951:Factor Xa