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• W2012700521 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 Arg323-Ile324, followed by cleavage at Arg274-Thr275. We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIaΔF1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIaΔF1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to Km. This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIaΔF1. 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 mIIaΔF1 to the enzyme. Specific recognition of mIIaΔF1 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. 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 Arg323-Ile324, followed by cleavage at Arg274-Thr275. We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIaΔF1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIaΔF1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to Km. This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIaΔF1. 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 mIIaΔF1 to the enzyme. Specific recognition of mIIaΔF1 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. meizothrombin acetothioacetyl adduct of FPR-CH2Cl dansyl-l-glutamyl-glycyl-l-arginine chloromethyl ketone 1,5-dimethylaminonaphthalene sulfonyl d-phenylalanyl-l-phenyl-alanyl-l-arginine chloromethyl ketone d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone thrombin inactivated with FPR-CH2Cl meizothrombin des fragment 1 mIIaΔF1 inactivated with DEGR-CH2Cl mIIaΔF1 inactivated with ATA-FPR-CH2Cl and modified with 6-(iodoacetamido)fluorescein following thioester hydrolysis mIIaΔF1 inactivated with FPR-CH2Cl H-d-phenylalanyl-l-pipecolyl-l-arginylp-nitroanilide methoxycarbonyl-d-cyclohexylglycyl-glycyl-l-arginylp-nitroanilide 4-aminobenzamidine small unilamellar vesicles composed of 75% (w/w) phosphatidylcholine and 25% (w/w) phosphatidylserine polyacrylamide gel electrophoresis Prothrombinase is an archetypal enzyme complex of blood coagulation (2Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (448) Google Scholar). 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 (2Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (448) Google Scholar, 3Mann K.G. Nesheim M.E. Church W.R. Haley P. Krishnaswamy S. Blood. 1990; 76: 1-16Crossref PubMed Google Scholar, 4Kalafatis M. Swords N.A. Rand M.D. Mann K.G. Biochim. Biophys. Acta. 1994; 1227: 113-129Crossref PubMed Scopus (105) Google Scholar). 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 (2Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (448) Google Scholar).Prothrombin is activated by proteolytic cleavage at two sites, Arg274-Thr275 and Arg323-Ile324, which yields the fragment 1.2 activation peptide and thrombin 1Residue numbers in prothrombin and derivatives are based on consecutive numbering of the 582 residues in the bovine zymogen. Thrombin consists of disulfide-linked A (residues 275–323) and B (residues 324–582) chains. Meizothombin consists of disulfide-linked fragment 1.2-A (residues 1–323) and B (residues 324–582) chains with intact Arg274-Thr275bond. Meizothrombin des fragment 1 consists of disulfide-linked fragment 2-A (residues 157–323) and B (residues 324–582) chains with intact Arg274-Thr275 bond. Prethrombin 2 consists of residues 275–582 with intact Arg323-Ile324 bond. Fragment 1.2 is residues 1–274. Fragment 2 is residues 157–274. Fragment 1 is residues 1–156. 1Residue numbers in prothrombin and derivatives are based on consecutive numbering of the 582 residues in the bovine zymogen. Thrombin consists of disulfide-linked A (residues 275–323) and B (residues 324–582) chains. Meizothombin consists of disulfide-linked fragment 1.2-A (residues 1–323) and B (residues 324–582) chains with intact Arg274-Thr275bond. Meizothrombin des fragment 1 consists of disulfide-linked fragment 2-A (residues 157–323) and B (residues 324–582) chains with intact Arg274-Thr275 bond. Prethrombin 2 consists of residues 275–582 with intact Arg323-Ile324 bond. Fragment 1.2 is residues 1–274. Fragment 2 is residues 157–274. Fragment 1 is residues 1–156. (5Owen W.G. Esmon C.T. Jackson C.M. J. Biol. Chem. 1974; 249: 594-605Abstract Full Text PDF PubMed Google Scholar, 6Downing M.R. Butkowski R.J. Clark M.M. Mann K.G. J. Biol. Chem. 1975; 250: 8897-8906Abstract Full Text PDF PubMed Google Scholar). The reaction catalyzed by prothrombinase proceeds almost exclusively via the initial cleavage at Arg323-Ile324, yielding meizothrombin as an intermediate, followed by cleavage at Arg274-Thr275 to yield the final products of the reaction (7Krishnaswamy S. Mann K.G. Nesheim M.E. J. Biol. Chem. 1986; 261: 8977-8984Abstract Full Text PDF PubMed Google Scholar, 8Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar). Single turnover kinetic studies indicate that the overall process is likely the sum of two consecutive enzyme-catalyzed reactions (8Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar). Consequently, steady state kinetic constants derived from measurements of the conversion of prothrombin to thrombin are difficult to interpret and are unlikely to provide valid mechanistic insights into this process. This problem can be circumvented by the use of proteolytic derivatives of prothrombin as analog substrates for the individual half-reactions of prothrombin activation (8Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar, 9Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 5386-5391Abstract Full Text PDF PubMed Google Scholar, 10Boskovic D.S. Giles A.R. Nesheim M.E. J. Biol. Chem. 1990; 265: 10497-10505Abstract Full Text PDF PubMed Google Scholar, 11Carlisle T.L. Bock P.E. Jackson C.M. J. Biol. Chem. 1990; 265: 22044-22055Abstract Full Text PDF PubMed Google Scholar).Prethrombin 2, generated by preparative cleavage at Arg274-Thr275, has been shown to be a valid substrate analog for kinetic studies of the cleavage at Arg323-Ile324, which represents the first cleavage reaction in the activation of prothrombin by prothrombinase (8Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar, 12Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar). Prior cleavage at Arg274-Thr275 was shown to have no effect on the recognition and cleavage of the Arg323-Ile324 site by prothrombinase (8Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar). Considerable advances in the understanding of substrate-prothrombinase interactions have been gained by studies with prethrombin 2 in the bovine system (12Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar, 13Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar, 14Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The results support a model in which the substrate, prethrombin 2, binds to prothrombinase via a multistep reaction (13Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar, 14Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The affinity of the enzyme for the substrate is determined by a bimolecular reaction between prethrombin 2 and extended macromolecular recognition sites (exosites) on the enzyme complex. This step is followed by interactions between elements surrounding the scissile bond with the active site of the enzyme followed by bond cleavage. The resulting product, thrombin, remains bound to the exosite and requires dissociation for subsequent rounds of catalysis. Since active site interactions between substrate and enzyme were found to be unfavorable, it has been suggested that binding specificity for cleavage at Arg323-Ile324 is largely determined by exosite interactions with the enzyme (13Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar).Comparable information on the second half-reaction, in which the Arg274-Thr275 peptide bond is cleaved by prothrombinase, is lacking. Although prior cleavage at Arg274-Thr275 does not influence the kinetics of cleavage at Arg323-Ile324, the reverse is not true (8Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar). Initial cleavage at Arg323-Ile324has been suggested to enhance the rate of cleavage at Arg274-Thr275 by a factor of ∼10 (11Carlisle T.L. Bock P.E. Jackson C.M. J. Biol. Chem. 1990; 265: 22044-22055Abstract Full Text PDF PubMed Google Scholar). The rate of cleavage at Arg274-Thr275, which converts meizothrombin to thrombin, is only modestly stimulated by factor Va (9Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 5386-5391Abstract Full Text PDF PubMed Google Scholar, 15Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar). Structural models based on x-ray diffraction studies indicate that the two cleavage sites in prothrombin are spatially distinct and separated by as much as 36 Å (16Martin P.D. Malkowski M.G. Box J. Esmon C.T. Edwards B.F. Structure. 1997; 5: 1681-1693Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 17Vijayalakshmi J. Padmanabhan K.P. Mann K.G. Tulinsky A. Protein Sci. 1994; 3: 2254-2271Crossref PubMed Scopus (151) Google Scholar). Finally, rapid kinetic studies support the possibility that the two cleavage reactions catalyzed by prothrombinase derive from two distinct types of substrate-enzyme interactions (8Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar). Taken together, these observations suggest that there may be significant differences in the mechanisms underlying the recognition and cleavage of the two bonds in prothrombin by prothrombinase.Meizothrombin (mIIa),2produced as the intermediate of prothrombin activation by prothrombinase following initial cleavage at Arg323-Ile324, is the relevant substrate analog for kinetic studies of the action of prothrombinase on the Arg274-Thr275 cleavage site (8Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar, 9Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 5386-5391Abstract Full Text PDF PubMed Google Scholar, 15Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar). Single turnover kinetic studies have established that proteolytic removal of the membrane-binding fragment 1 domain from mIIa to yield meizothrombin des fragment 1 (mIIaΔF1) does not affect the kinetics of substrate recognition and cleavage by prothrombinase (8Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar). Thus, although mIIa can bind membranes (18Armstrong S.A. Husten E.J. Esmon C.T. Johnson A.E. J. Biol. Chem. 1990; 265: 6210-6218Abstract Full Text PDF PubMed Google Scholar, 19Pei G. Laue T.M. Aulabaugh A. Fowlkes D.M. Lentz B.R. Biochemistry. 1992; 31: 6990-6996Crossref PubMed Scopus (7) Google Scholar), this interaction does not play an obvious enhancing role in its ability to be recognized and cleaved by prothrombinase. Therefore, mIIaΔF1 is a valid substrate analog for the second half-reaction of prothrombin activation that permits studies of the enzyme-substrate interaction in the absence of the obscuring effects of membrane-mediated substrate delivery steps (8Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar, 20Nelsestuen G.L. Martinez M.B. Biochemistry. 1997; 36: 9081-9086Crossref PubMed Scopus (28) Google Scholar, 21Lu Y. Nelsestuen G.L. Biochemistry. 1996; 35: 8201-8209Crossref PubMed Scopus (22) Google Scholar). We have therefore pursued steady state kinetic studies of the cleavage of bovine mIIaΔF1 by bovine prothrombinase to further investigate the mechanisms underlying macromolecular substrate recognition by prothrombinase.DISCUSSIONEvidence for a major role of exosite interactions in the productive pathway for protein substrate recognition by prothrombinase has been previously developed in kinetic studies using prethrombin 2 as a substrate analog for cleavage at the Arg323-Ile324 peptide bond in prothrombin (13Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar, 14Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Data obtained in the present work using mIIaΔF1 suggest that equivalent exosite interactions are relevant for protein substrate recognition and cleavage at the Arg274-Thr275site as well.Evidence to support this conclusion derives from the observation that the product, thrombin (IIai), acts as a competitive inhibitor of the cleavage of either prethrombin 2 (Arg323-Ile324 cleavage) or mIIaΔF1 (Arg274-Thr275 cleavage) by prothrombinase. In contrast, the binding of IIai to prothrombinase has no obvious effect on the access of small ligands and peptidyl substrates to the active site of Xa within prothrombinase (13Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar, 14Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Competitive inhibition of prethrombin 2 activation by IIai occurs with a Ki comparable to the affinity of prothrombinase for the substrate (13Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar). In the case of mIIaΔF1 cleavage, theKi for IIai is exactly equal to the apparent affinity of the enzyme for this substrate. Thrombin and prethrombin 2 share a series of structural features (17Vijayalakshmi J. Padmanabhan K.P. Mann K.G. Tulinsky A. Protein Sci. 1994; 3: 2254-2271Crossref PubMed Scopus (151) Google Scholar), while thrombin represents the COOH-terminal domain of mIIaΔF1 (16Martin P.D. Malkowski M.G. Box J. Esmon C.T. Edwards B.F. Structure. 1997; 5: 1681-1693Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). These points justify the reasonable conclusion that equivalent interactions with an enzymic exosite underlie the recognition and cleavage of both analog substrates for the two half-reactions of prothrombin activation.Alternate substrate studies with mIIaΔF1 have permitted a resolution of the thermodynamic contributions of the presumed exosite (Ks, Scheme FSI) and active site interactions (Ks*, Scheme FSI) to substrate affinity. Although the overall affinity of prothrombinase for mIIaΔF1 is equal to the affinity for IIai (i.e. Km =Kp), this equivalence does not apply to the inferred thermodynamics of the exosite-binding step. The inferred equilibrium dissociation constant for the binding of mIIaΔF1 to the exosite (Ks ≅ 12 μm) is approximately 4-fold greater than the values determined for exosite binding by prethrombin 2 or IIai (13Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar, 14Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). However, mIIaΔF1 contains the fragment 2 domain that is established to modulate interactions between the protease domain of the substrate and other macromolecular ligands (51Liu L.W. Ye J. Johnson A.E. Esmon C.T. J. Biol. Chem. 1991; 266: 23633-23636Abstract Full Text PDF PubMed Google Scholar, 52Bock P.E. Olson S.T. Bjork I. J. Biol. Chem. 1997; 272: 19837-19845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 53Fredenburgh J.C. Stafford A.R. Weitz J.I. J. Biol. Chem. 1997; 272: 25493-25499Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Thus, the data obtained in studies of mIIaΔF1 cleavage require comparison with the kinetics of activation of prethrombin 2 saturated with fragment 2. The value forKs inferred for mIIaΔF1 in the present work is strikingly similar to the Km previously determined for prethrombin 2 plus fragment 2 (12Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar). For prethrombin 2 activation, the data are consistent with an unfavorable active site docking step, implying Ks* ≫ 1 (estimated by simulations at Ks* ≥ 8) andKm ≅ Ks in substrate binding steps equivalent to those illustrated in Scheme FSI (13Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar).The present results, and the application of Equations 4 and 5 to the steady state kinetic constants for prethrombin 2 plusfragment 2 (12Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar), allow for a more appropriate comparison of the stepwise binding interactions that lead to the recognition of the two bonds in the substrate by prothrombinase followed by catalysis (SchemeFSII). Although prethrombin 2plus fragment 2 and mIIaΔF1 are individually cleaved by prothrombinase with different steady state kinetic constants, the dissociation constant for the exosite binding step (Ks) and the inferred rate constant for catalysis (kcat) for these two substrates are equivalent (Scheme FSII). The major difference appears to lie in the equilibrium dissociation constant for the active site docking step (Ks*) that precedes bond cleavage. Active site interactions that precede the cleavage of the Arg274-Thr275 site in mIIaΔF1 appear to be modestly favorable, whereas the comparable binding step that precedes cleavage of the Arg323-Ile324 site in prethrombin 2 plus fragment 2 is an unfavorable step (SchemeFSII).Comparable values for Ks inferred for both protein substrate analogs (Scheme FSII) supports the contention that the initial interaction between both substrates and prothrombinase involves equivalent exosite binding steps. Since prethrombin 2 and thrombin appear to bind this enzymic exosite with greater affinity (Ks ≅ 3 μm) (13Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar, 14Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), it follows that the relatively high affinity interaction between fragment 2 and prethrombin 2 (12Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar) modestly decreases the affinity of the resultant substrate for the enzymic exosite by a factor of 4. The binding of fragment 2 to prethrombin 2 has also been established to increase theVmax for substrate cleavage by prothrombinase by approximately the same factor (12Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar). Thus, while the rate-enhancing effects of fragment 2 on the cleavage of prethrombin 2 by prothrombinase are well established in the literature (12Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar, 54Bajaj S.P. Butkowski R.J. Mann K.G. J. Biol. Chem. 1975; 250: 2150-2156Abstract Full Text PDF PubMed Google Scholar, 55Esmon C.T. Jackson C.M. J. Biol. Chem. 1974; 249: 7791-7797Abstract Full Text PDF PubMed Google Scholar), the data are most consistent with the interpretation that the binding of fragment 2 to prethrombin 2 somehow alters the structure of the substrate, leading to a modest perturbation in the kinetic constants (12Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar). Based on previous studies with proteolytic fragments of prethrombin 2 and thrombin (14Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), it seems probable that the reduced affinity of the fragment 2-prethrombin 2 complex for exosite binding to prothrombinase arises from linkage between distinct sites in the proteinase domain of the substrate that mediate fragment 2 binding and interactions with the enzymic exosite.The interaction of fragment 2 with thrombin is also likely to reduce the affinity of the product for exosite binding. If the affinity changes observed with prethrombin 2 directly apply to thrombin, it follows that the equilibrium dissociation constant for the interaction between thrombin and the enzymic exosite is approximately 4-fold lower than that of the fragment 2-thrombin complex i.e. Kp ≅ 3 μm, βKp ≅ 12 μm, β ≅ 4 (SchemeFSI). Consequently, the previously measured equilibrium dissociation constant for the binding of fragment 2 to thrombin (Kd = 5 μm) (46Bock P.E. J. Biol. Chem. 1992; 267: 14974-14981Abstract Full Text PDF PubMed Google Scholar) is also expected to be increased by a factor of 4 in the presence of prothrombinasei.e. KF2,IIa ≅ 5 μm, βKF2,IIa ≅ 20 μm (Scheme FSI). These points provide a reasonable quantitative accounting for the inhibition of mIIaΔF1 cleavage by IIai as well as the modest increase in reaction rate observed in the presence of increasing concentrations of fragment 2 (Fig. 6).Evidence for a high affinity interaction between fragment 2 and thrombin (Kd = 0.8 nm) has previously been obtained at an ionic strength much lower than those used in the present work (56Myrmel K.H. Lundblad R.L. Mann K.G. Biochemistry. 1976; 15: 1767-1773Crossref PubMed Scopus (48) Google Scholar). A strong ionic strength dependence of this interaction is implied by the substantially larger equilibrium dissociation constant measured by Bock at I = 0.15 m(46Bock P.E. J. Biol. Chem. 1992; 267: 14974-14981Abstract Full Text PDF PubMed Google Scholar), which seems to represent the most appropriate value for considerations of product inhibition in the present studies. However, a value of KF2,IIa substantially lower than 5 μm would provide more compelling support for the conclusion that fragment 2 binding does not enhance the ability of IIai to bind prothrombinase and would imply a far stronger destabilizing effect of fragment 2 on this interaction.Significant differences in the rate constant for catalysis, inferred by division of Vmax by ET, have been previously noted for the two cleavage reactions in the protein substrate catalyzed by prothrombinase (9Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 5386-5391Abstract Full Text PDF PubMed Google Scholar, 10Boskovic D.S. Giles A.R. Nesheim M.E. J. Biol. Chem. 1990; 265: 10497-10505Abstract Full Text PDF PubMed Google Scholar). An obvious explanation for this finding has not been forthcoming since identical P1–P4 residues precede both cleavage sites in prothrombin (36Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (96) Google Scholar). Assuming that Scheme FSII provides an adequate description of the binding steps in substrate recognition, the present results indicate that the rate constant for catalysis is essentially the same for the two cleavage reactions and is comparable to values observed for the cleavage of peptidyl substrates bearing the same P1–P4 sequence encountered in the protein substrate (57Lottenberg R. Hall J.A. Pautler E. Zupan A. Christensen U. Jackson C.M. Biochim. Biophys. Acta. 1986; 874: 326-336Crossref PubMed Scopus (30) Google Scholar).Structural models for mIIaΔF1 and prethrombin 2 from x-ray diffraction data indicate that the residues preceding the Arg323-Ile324 bond are either disordered or require significant rearrangement to be successfully docked into the active site of factor Xa (17Vijayalakshmi J. Padmanabhan K.P. Mann K.G. Tulinsky A. Protein Sci. 1994; 3: 2254-2271Crossref PubMed Scopus (151) Google Scholar). Such features are not observed for the identical residues preceding the Arg274-Thr275bond in mIIaΔF1 (16Martin P.D. Malkowski M.G. Box J. Esmon C.T. Edwards B.F. Structure. 1997; 5: 1681-1693Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). These observations may provide a structural explanation for the large differences in the equilibrium constant for the active site docking step (Ks*, Scheme FSII) inferred for the two protein substrates. Previous work has established that, although cleavage at Arg323-Ile324 in prethrombin 2 is greatly accelerated by factor Va, the cofactor has a much smaller effect on the cleavage at the Arg274-Thr275 bond in mIIaΔF1 (9Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 5386-5391Abstract Full Text PDF PubMed Google Scholar, 15Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar). If it is indeed true that exosite binding by the protein substrate is only significant following assembly of the prothrombinase complex (13Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar, 50Betz A. Vlasuk G.P. Bergum P.W. Krishnaswamy S. Biochemistry. 1997; 36: 181-191Crossref PubMed Scopus (31) Google Scholar, 58Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), then this initial interaction, which serves to tether the substrate to the enzyme complex, is likely to disproportionately enhance cleavage at the disordered Arg323-Ile324 site governed by an unfavorable active site docking step in contrast to cleavage at the Arg274-Thr275 site, which results from a favorable interaction at the active site. This hypothesis implies that the rate-enhancing effects of factor Va at least partly arise from indirect or direct contributions toward exosite binding by the protein substrate. This initial tethering reaction could overcome inefficient catalysis at suboptimally configured cleavage sites in the protein substrate.Structural studies of prothrombin derivatives indicate that the two scissile bonds in the substrate are separated by as much as 36 Å (16Martin P.D. Malkowski M.G. Box J. Esmon C.T. Edwards B.F. Structure. 1997; 5: 1681-1693Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), yet the present data indicate that both cleavages derive from equivalent exosite interactions that initially tether the substrate to" @default.
• W2012700521 created "2016-06-24" @default.
• W2012700521 creator A5033304595 @default.
• W2012700521 creator A5036209779 @default.
• W2012700521 date "2000-12-01" @default.
• W2012700521 modified "2023-10-17" @default.
• W2012700521 title "Exosite Binding Tethers the Macromolecular Substrate to the Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct Sites" @default.
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