Abstract
The preferred pathway for prothrombin activation by prothrombinase involves initial cleavage at Arg(320) to produce meizothrombin, which is then cleaved at Arg(271) to liberate thrombin. Exosite binding drives substrate affinity and is independent of the bond being cleaved. The pathway for cleavage is determined by large differences in V(max) for cleavage at the two sites within intact prothrombin. By fluorescence binding studies in the absence of catalysis, we have assessed the ability of the individual cleavage sites to engage the active site of Xa within prothrombinase at equilibrium. Using a panel of recombinant cleavage site mutants, we show that in intact prothrombin, the Arg(320) site effectively engages the active site in a 1:1 interaction between substrate and enzyme. In contrast, the Arg(271) site binds to the active site poorly in an interaction that is approximately 600-fold weaker. Perceived substrate affinity is independent of active site engagement by either cleavage site. We further show that prior cleavage at the 320 site or the stabilization of the uncleaved zymogen in a proteinase-like state facilitates efficient docking of Arg(271) at the active site of prothrombinase. Therefore, we establish direct relationships between docking of either cleavage site at the active site of the catalyst, the V(max) for cleavage at that site, substrate conformation, and the resulting pathway for prothrombin cleavage. Exosite tethering of the substrate in either the zymogen or proteinase conformation dictates which cleavage site can engage the active site of the catalyst and enforces the sequential cleavage of prothrombin by prothrombinase.
Highlights
Thrombin, the key effector product of the coagulation cascade, is produced by specific proteolysis of the zymogen precursor, prothrombin [1]
The use of XaS195A and fluorescent S1 site probe, pAB, has permitted the investigation of the ability of prothrombin and its derivatives to engage the active site of factor Xa within prothrombinase but in the absence of catalysis
Structures flanking the Arg320 cleavage site can effectively bind the active site of prothrombinase and displace pAB
Summary
Our approach employs equilibrium binding measurements in the absence of catalysis to provide a model-independent assessment of the ability of the individual cleavage site prothrombin derivatives to dock at the active site of factor Xa within prothrombinase. The rationale for using PCPSLUV in activity measurements at subnanomolar concentrations of enzyme has been described [12] This approach permits the use of saturating concentrations of phospholipid yet minimizes kinetic complexity associated with non-productive binding of substrate to vesicles lacking enzyme [12]. Fitting according to Scheme II with these constraints provided estimates for Ks* and the fluorescence signals for free and enzyme-bound P normalized on a molar basis Division of these two fluorescence yields along with the propagation of the individual errors provided a fitted value for ⌬FMAX/FFree equivalent to the term derived from the pAB binding studies. We have deliberately qualified fitted values of Ks* as upper or lower limit estimates without ascribing significance to fitted confidence limits in this parameter
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