[23] Prothrombin

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...

Section: Discussionmentioning

confidence: 51%

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

Boskovic

Krishnaswamy²

2000

“…Factors Xa and Va were prepared and characterized as described before (20,24). The derivatives F12, F2, P2, mIIa, and IIa were prepared by preparative cleavage of prothrombin purified from plasma (II PL ) and subsequent repurification using established procedures (25). An equivalent strategy using II A195 as substrate was employed to generate IIa A195 and P2 A195 .…”

Section: Methodsmentioning

confidence: 99%

“…Inactivated derivatives of IIa and mIIa (IIai and mIIai) were prepared by covalent modification with FPRck as described (12). Protein concentrations were determined using the following molecular weights and extinction coefficients (E 280 (25). Prothrombin variants were exchanged into assay buffer by centrifugal gel filtration before use.…”

Section: Methodsmentioning

confidence: 99%

“…Cleavage at Arg 155 -Both thrombin and mIIa are well known to catalyze cleavage at Arg 155 in prothrombin and its derivatives (25,39,40). Thus, high concentrations of proteinase produced during prothrombin activation necessitate the use of thrombin inhibitors such as DAPA to selectively inhibit this feedback reaction (41).…”

Section: Membrane-bound Species During Prothrombin Activationmentioning

confidence: 99%

See 1 more Smart Citation

Fate of Membrane-bound Reactants and Products during the Activation of Human Prothrombin by Prothrombinase

Kamath

Krishnaswamy

2008

Membrane binding by prothrombin, mediated by its N-terminal fragment 1 (F1) domain, plays an essential role in its proteolytic activation by prothrombinase. Thrombin is produced in two cleavage reactions. One at Arg 320 yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg 271 , yields thrombin and severs covalent linkage with the N-terminal fragment 1.2 (F12) region. Covalent linkage with the membrane binding domain is also lost when prethrombin 2 (P2) and F12 are produced following initial cleavage at Arg 271 . We show that at the physiological concentration of prothrombin, thrombin formation results in rapid release of the proteinase into solution. Product release from the surface can be explained by the weak interaction between the proteinase and F12 domains. In contrast, the zymogen intermediate P2, formed following cleavage at Arg 271 , accumulates on the surface because of a ϳ20-fold higher affinity for F12. By kinetic studies, we show that this enhanced binding adequately explains the ability of unexpectedly low concentrations of F12 to greatly enhance the conversion of P2 to thrombin. Thus, the utilization of all three possible substrate species by prothrombinase is regulated by their ability to bind membranes regardless of whether covalent linkage to the F12 region is maintained. The product, thrombin, interacts with sufficiently poor affinity with F12 so that it is rapidly released from its site of production to participate in its numerous hemostatic functions.Thrombin, the key effector serine proteinase of the blood coagulation cascade, is produced by specific and limited proteolysis of the zymogen, prothrombin. The physiologically relevant catalyst for this reaction is the prothrombinase complex consisting of the serine proteinase, factor Xa, and the cofactor, factor Va, assembled on membranes in the presence of Ca 2ϩ(1). In addition to facilitating the assembly of the enzyme complex, membranes containing acidic or amino phospholipids play an important role in mediating the delivery of prothrombin to the membrane-bound enzyme (1, 2). This arises from the ability of prothrombin to bind to these membranes through the fragment 1 (F1) 2 domain present at its N terminus (1, 3, 4). Thrombin, derived from the C-terminal half of prothrombin, is produced as a result of cleavages 3 following Arg 271 and Arg 320(1, 3, 5). Cleavage at Arg 320 converts the zymogen to a proteinase, whereas cleavage at Arg 271 severs covalent linkage with the N-terminal fragment 1.2 (F12) domain harboring the membrane binding site (Scheme 1). Covalent linkage of the C-terminal domain with F12 is also lost in the zymogen intermediate, prethrombin 2 (P2), produced following cleavage only at Arg 271 (Scheme 1). In contrast, meizothrombin (mIIa), produced following cleavage only at Arg 320 is covalently linked to the membrane binding domain through a disulfide bond (Scheme 1). Accordingly, both prothrombin and mIIa are established to bind to membranes, and this binding interaction impacts the...

“…Prothrombin is activated to thrombin by two factor Xa-catalyzed cleavages, one at Arg-320 and the other at Arg-271 (5,6). Because two cleavages are required for activation, two intermediates are possible.…”

mentioning

confidence: 99%

Further Evidence for Two Functional Forms of Prothrombinase Each Specific for Either of the Two Prothrombin Activation Cleavages

Kim

Nesheim

2007

Self Cite

Previous work showed that prothrombin derivatives cleavable only at Arg-320 (rMZ) or Arg-271 (rP2) are partial, rather than competitive, inhibitors of prothrombin activation by prothrombinase. A "ping-pong"-like model, which posits two equilibrating forms of prothrombinase, explained the inhibition pattern. The present studies were undertaken to further investigate this putative mechanism. Two models were developed, one allowing for one form of the enzyme and the other allowing for two forms. Both models also allowed channeling and ratcheting. The models were fit to full time courses of prothrombin, meizothrombin, prethrombin-2, and the B-chain. In the absence of ratcheting and channeling, neither model fits the data. In their presence, however, both models fit very well, and thus they could not be distinguished. Therefore, inhibition of rMZ activation by rP2 was studied. Inhibition was partial and the two-form model fit the data with randomly distributed residuals, whereas the oneform model did not. Initial rates of fluorescein-labeled prothrombin cleavage in the presence of various prothrombin derivatives reported by Brufatto and Nesheim (Brufatto, N., and Nesheim, M. E. (2003) J. Biol. Chem. 278, 6755-6764) were also analyzed using the two models. The two-form model fit the partial inhibition data well, whereas the one-form model did not. In addition, prothrombin at varying concentrations was activated, and subsequently, the initial rates were plotted with respect to the initial prothrombin concentration. When compared with the expected initial rates as determined by the simulation of the models, the two-form model fit the observed rates better than the one-form model. The results obtained here further support the existence of two functional forms of prothrombinase.