Prothrombinase, the membrane bound complex of factors Xa and Va, catalyzes thrombin formation by the cleavage of prothrombin at R320 followed by R271. The two cleavage sites are expected to be ~40Å apart in prothrombin. Essentially ordered cleavage arises from exosite tethering of prothrombin to the enzyme which facilitates engagement of the R320 site but precludes docking of the R271site to the active site of the catalyst. Prior cleavage at R320 appears necessary for efficient active site engagement by the R271 site. The physical bases for these observations have been investigated using fluorescence resonance energy transfer to assess the disposition of the 271 site to the active site of the catalyst. Expression and purification of a prothrombin variant containing C in place of R271 (IIC271) surprisingly yielded a product lacking free cysteines. A free thiol could be rescued following treatment with reduced glutathione and could be modified with PEG or Alexa532 maleimides yielding ~1 mol reagent incorporated /mol protein. Characterization of IIC271 modified with Alexa532 maleimide (IIC271*) established that the probe was covalently contained only in those fragments bearing residue 271. IIC271* could be cleaved normally by prothrombinase at R320 yielding a product with the expected catalytic activity. Thus, thiol rescue permits selective modification of the engineered 271 site without obvious deleterious effects. Fluorescence studies employed prothrombinase assembled using Xa containing Alexa488 covalently linked to the active site with a peptidyl chloromethylketone as donor and IIC271* as acceptor. Increasing concentrations of IIC271* yielded a large and saturable decrease in donor fluorescence. This decrease could be reversed by the addition of excess unlabelled prothrombin, was not observed in the absence of membranes and could be eliminated by the addition of EDTA. Changes in donor fluorescence exhibit the features expected for energy transfer arising from the binding of prothrombin to prothrombinase. Titrations with increasing concentrations of IIC271* yielded Kd = 40.7 ± 13.5 nM and n = 1.12 ± 0.23 mol IIC271*/mol prothrombinase. Agreement between the Kd measured using active site blocked enzyme and the previously measured Km further substantiates the primary role played by exosite binding in determining substrate affinity. Maximal energy transfer efficiency inferred from donor quenching (79.3 ± 1.7%) was confirmed by measurements of excited state lifetime. Based on the measured Forster distance (57.4 Å), this value indicates that the acceptor at the 271 site in IIC271* is ~46 Å away from the donor at the active site of Xa within prothrombinase. IIC271* was cleaved by ecarin at R320 to yield meizothrombin (mIIaC271*), stabilized by covalent inactivation and re-purified. Similar experiments performed with mIIaC271* as acceptor yielded substantial differences in the extent of donor quenching in comparison to IIC271*. Thus, major changes are evident at the 271 site following cleavage at R320. We conclude that exosite-dependent binding of prothrombin to prothrombinase constrains the substrate such that the 271 site is distant from and cannot efficiently engage the active site of the catalyst. This explains the inability of prothrombinase to efficiently cleave at R271 in prothrombin. Proteinase formation following initial cleavage at R320 reorients the 271 site with respect to the active site of prothrombinase. Our findings offer a physical explanation for how prothrombinase is able to discriminate between the cleavage sites in prothrombin resulting in the ordered cleavage of the two distant sites within the substrate.

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