The isomorphous structures of prethrombin2, hirugen–, and PPACK–thrombin: Changes accompanying activation and exosite binding to thrombin

Vijayalakshmi, J.; Padmanabhan, K.; Mann, Kenneth G.; Tulinsky, A.

doi:10.1002/pro.5560031211

Cited by 158 publications

(230 citation statements)

References 65 publications

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“…In the case of mIIa⌬F1 cleavage, the K i for IIa i is exactly equal to the apparent affinity of the enzyme for this substrate. Thrombin and prethrombin 2 share a series of structural features (17), while thrombin represents the COOHterminal domain of mIIa⌬F1 (16). 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.…”

Section: Discussionmentioning

confidence: 75%

“…Structural models for mIIa⌬F1 and prethrombin 2 from xray diffraction data indicate that the residues preceding the Arg 323 -Ile 324 bond are either disordered or require significant rearrangement to be successfully docked into the active site of factor Xa (17). Such features are not observed for the identical residues preceding the Arg 274 -Thr 275 bond in mIIa⌬F1 (16).…”

Section: Discussionmentioning

confidence: 99%

“…Meizothombin consists of disulfide-linked fragment 1.2-A (residues (9,15). 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 (16,17). Finally, rapid kinetic studies support the possibility that the two cleavage reactions catalyzed by prothrombinase derive from two distinct types of substrate-enzyme interactions (8).…”

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confidence: 99%

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

Boskovic

Krishnaswamy²

2000

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Boskovic

Krishnaswamy²

2000

Journal of Biological Chemistry

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“…In addition to the allosteric effect of Na ϩ , thrombin activity and specificity is influenced allosterically by binding of ligands to exosite I (24-28), a domain located 25-Å away from the active site and on the opposite pole of the molecule relative to the Na ϩ site (16, 29). The exact mechanism of this long-range communication remains controversial (30,31). A recent structure of murine thrombin bound to a fragment of PAR3 at exosite I has revealed a significant conformational change of the 60-loop that opens the active site fully for optimal substrate diffusion (32).…”

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confidence: 99%

Structural identification of the pathway of long-range communication in an allosteric enzyme

Gandhi

Chen

Mathews

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

103

142

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Allostery is a common mechanism of regulation of enzyme activity and specificity, and its signatures are readily identified from functional studies. For many allosteric systems, structural evidence exists of long-range communication among protein domains, but rarely has this communication been traced to a detailed pathway. The thrombin mutant D102N is stabilized in a self-inhibited conformation where access to the active site is occluded by a collapse of the entire 215-219 ␤-strand. Binding of a fragment of the protease activated receptor PAR1 to exosite I, 30-Å away from the active site region, causes a large conformational change that corrects the position of the 215-219 ␤-strand and restores access to the active site. The crystal structure of the thrombin-PAR1 complex, solved at 2.2-Å resolution, reveals the details of this long-range allosteric communication in terms of a network of polar interactions.protease activated receptor ͉ thrombin ͉ x-ray crystallography E ver since its original formulation (1, 2), the allosteric concept of enzyme regulation has captivated the interest of structural biologists. The idea that events occurring at a given site of the protein can be transmitted long-range to affect affinity or catalytic efficiency at a distant site offers an elegant explanation for linkage and cooperativity (3) but poses a challenge to the crystallographer who seeks to identify the communication pathway underlying the functional effects. Perutz (4) was the first to offer a molecular explanation for the allosteric mechanism of hemoglobin cooperativity. For multimeric proteins like hemoglobin, conformational transitions tend to affect the quaternary structure and signatures of allostery have been detected crystallographically (5-9). Allostery is not limited to multimeric assemblies and, in fact, many monomeric proteins feature conformational plasticity that translate into allosteric behavior at equilibrium and steady state (6, 10, 11). For such systems, the structural signatures of long-range communication tend to manifest themselves as small changes in tertiary structure or Hbonding connectivity (12, 13) and lack the amplification seen in large quaternary structural reorganization. Identification of the pathway of communication underlying an allosteric mechanism remains a difficult task in general and continues to receive utmost attention (11, 13). Alternative approaches have been proposed to identify such pathways based on the statistical analysis of evolutionary records of protein sequences (14) or anisotropic thermal diffusion (15). Although promising and insightful, such approaches must ultimately find validation by structural investigation.Among Na ϩ -activated allosteric enzymes (6), the serine protease thrombin has received much attention in view of its critical roles in hemostasis and thrombosis (12,16,17). Thrombin features considerable structural plasticity and exists predominantly in two forms at equilibrium, the Na ϩ -free slow form E (Ϸ40% of the molecules in vivo) and the Na ϩ -bound fast f...

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“…In addition to binding its receptor, proteolytic substrates, and several physiological inhibitors, thrombin also binds exogenous inhibitors such as hirudin (produced as an anticoagulant by leeches) and a substrate transitionstate analog, D-Phe-Pro-Arg chloromethyl ketone (PPACK). Thrombin contains two major ligand binding sites: the active site and the fibrinogen binding site, or exosite, which provides an additional binding surface, enhancing the affinity for the fibrinogen substrate and hirudin and its analogs (Vijayalakshmi et al, 1994). The variety of crystallographic thrombin:ligand complexes provides a basis for studying water sites that are conserved in thrombin regardless of ligand, as well as those water sites that are ligand-specific.…”

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confidence: 99%

Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity

Sanschagrin

Kuhn

1998

Protein Science

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Cluster analysis is presented as a technique for analyzing the conservation and chemistry of water sites from independent protein structures, and applied to thrombin, trypsin, and bovine pancreatic trypsin inhibitor (BPTI) to locate shared water sites, as well as those contributing to specificity. When several protein structures are superimposed, complete linkage cluster analysis provides an objective technique for resolving the continuum of overlaps between water sites into a set of maximally dense microclusters of overlapping water molecules, and also avoids reliance on any one structure as a reference. Water sites were clustered for ten superimposed thrombin structures. three trypsin structures, and four BPTI structures. For thrombin, 19% of the 708 microclusters, representing unique water sites, contained water molecules from at least half of the structures, and 4% contained waters from all 10. For trypsin, 77% of the 106 microclusters contained water sites from at least half of the structures, and 57% contained waters from all three. Water site conservation correlated with several environmental features: highly conserved microclusters generally had more protein atom neighbors, were in a more hydrophilic environment, made more hydrogen bonds to the protein, and were less mobile. There were significant overlaps between thrombin and trypsin conserved water sites, which did not localize to their similar active sites, but were concentrated in buried regions including the solvent channel surrounding the Na' site in thrombin, which is associated with ligand selectivity. Cluster analysis also identified water sites conserved in thrombin but not trypsin, and vice versa, providing a list of water sites that may contribute to ligand discrimination. Thus, in addition to facilitating the analysis of water sites from multiple structures, cluster analysis provides a useful tool for distinguishing between conserved features within a protein family and those conferring specificity.

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The isomorphous structures of prethrombin2, hirugen–, and PPACK–thrombin: Changes accompanying activation and exosite binding to thrombin

Cited by 158 publications

References 65 publications

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

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

Structural identification of the pathway of long-range communication in an allosteric enzyme

Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity

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