Exosite Binding Tethers the Macromolecular Substrate to the Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct Sites

Abstract

The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg 323 -Ile 324 , followed by cleavage at Arg 274 -Thr 275 . We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIa⌬F1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIa⌬F1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to K m . This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIa⌬F1. As thrombin is known to bind to an exosite on prothrombinase, initial interactions at an exosite likely play a role in the enzyme-substrate interaction. Occupation of the active site of prothrombinase by a reversible inhibitor does not exclude the binding of mIIa⌬F1 to the enzyme. Specific recognition of mIIa⌬F1 is achieved through an initial bimolecular reaction with an enzymic exosite, followed by an active site docking step in an intramolecular reaction prior to bond cleavage. By alternate substrate studies, we have resolved the contributions of the individual binding steps to substrate affinity and catalysis. This pathway for substrate binding is identical to that previously determined with a substrate analog for the first half-reaction of prothrombin activation. We show that differences in the observed kinetic constants for the two cleavage reactions arise entirely from differences in the inferred equilibrium constant for the intramolecular binding step that permits elements surrounding the scissile bond to dock at the active site of prothrombinase. Therefore, substrate specificity is achieved by binding interactions with an enzymic exosite that tethers the protein substrate to prothrombinase and directs cleavage at two spatially distinct scissile bonds.Prothrombinase is an archetypal enzyme complex of blood coagulation (2). The enzyme complex assembles through well characterized, reversible, protein-protein and protein-membrane interactions between the serine protease, factor Xa, the cofactor, factor Va, and membranes in the presence of calcium ions (2-4). The resulting complex catalyzes the conversion of prothrombin to thrombin at a greatly enhanced rate, compared with the reaction rate catalyzed by factor Xa alone (2).Prothrombin is activated by proteolytic cleavage at two sites, Arg 274 -Thr 275 and Arg 323 -Ile 324 , which yields the fragment 1.2 activation peptide and thrombin 1 (5, 6). The reaction catalyzed by prothrombinase proceeds almost exclusively via the initial cleavage at Arg 323 -Ile 324 , yielding meizothrombin as an intermediate, followed by cleavage at Arg 274 -Thr 275 to yield the final products of the reaction (7,8). Single turnover kinetic studies indicate that the overall process is likely the sum of two consecutive enzyme-catalyzed reactions (8). Consequently, steady state...

Binding of Substrate in Two Conformations to Human Prothrombinase Drives Consecutive Cleavage at Two Sites in Prothrombin

“…Proteolytic derivatives of human prothrombin (II PL ), fragment 1.2 (F1.2), prethrombin 2 (P2), and thrombin were purified and characterized by established procedures (5). Ecarin was purified from the venom of Echis carinatus pyramidum (Latoxan, Valence, France) (19). Modifications to procedures developed with bovine prothrombin were employed to prepare and purify human mIIa covalently inactivated with FPR-CH 2 Cl (mIIa i ) and mIIa inactivated with ATA-FPR-CH 2 Cl and labeled with 6-(iodoacetamido)fluorescein (mIIa F ) (17,19).…”

“…Ecarin was purified from the venom of Echis carinatus pyramidum (Latoxan, Valence, France) (19). Modifications to procedures developed with bovine prothrombin were employed to prepare and purify human mIIa covalently inactivated with FPR-CH 2 Cl (mIIa i ) and mIIa inactivated with ATA-FPR-CH 2 Cl and labeled with 6-(iodoacetamido)fluorescein (mIIa F ) (17,19). Following purification, both mIIa i and mIIa F were dialyzed into 20 mM Hepes, 0.15 M NaCl, pH 7.5, concentrated by ultrafiltration, and stored at Ϫ20°C.…”

“…The kinetics of cleavage at Arg 271 * in mIIa was inferred from continuous measurements of this fluorescence change using a strategy previously established in the bovine system (19). Measurements were performed in a SpectraMax Gemini (Molecular Devices, Sunnyvale, CA) kinetic plate reader using black polystyrene 96-well plates that had been pretreated with 0.1% (v/v) Tween 20 in Assay Buffer and air-dried before use.…”

Nematode Anticoagulant Protein c2 Reveals a Site on Factor Xa That Is Important for Macromolecular Substrate Binding to Human Prothrombinase

“…The initial exosite-binding step dominates the affinity and binding specificity of the enzyme for the protein substrate. This interaction serves to tether the substrate to the enzyme, thereby directing ordered active-site docking and cleavage at two spatially distinct sites in the zymogen (11). Thus, surfaces on the enzyme complex, removed from the active site of factor Xa, contribute in a dominant way to the productive recognition of the protein substrate.…”

The Elusive Role of the Potential Factor X Cation-binding Exosite-1 in Substrate and Inhibitor Interactions

“…A number of studies have established that the specific cleavage of the two peptide bonds in prothrombin by prothrombinase is governed by interactions remote from the catalytic groove (13,40). To explore if the potential cation-binding exosite-1 of FXa is important in prothrombin activation, we recorded progress curves of pNA release and evaluated the acceleration of S2238 hydrolysis due to increasing thrombin concentrations (Fig.…”