Human .alpha.- to .zeta.-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained

Brezniak, Diane V.; Brower, Mark S.; Witting, Joyce I.; Walz, Daniel A.; Fenton, John W.

doi:10.1021/bi00466a017

Cited by 56 publications

(41 citation statements)

References 31 publications

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“…The salt bridge between h-Asp-55 and Lys-149E may not make a large contribution to binding energy. Lys-149E is found in a loop in thrombin that assumes a different conformation in the D-PhePro-Arg-CH2-thrombin complex (Bode et al, 1989;Grutter et al, 1990;Rydel et al, 1990), and the susceptibility of this loop to cleavage by proteinases suggests that it is mobile (Berliner, 1984;Kawabata et al, 1985;Brezniak et al, 1990 (Carter et al, 1984;Ackers & Smith, 1985;Wells, 1990): Wells (1990) has applied equations of the same form as eqn.…”

Section: Resultsmentioning

confidence: 99%

Ionic interactions in the formation of the thrombin-hirudin complex

Betz

Hofsteenge

Stone

1991

Biochemical Journal

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Site-directed mutagenesis has been used to examine the importance of each of the acidic C-terminal residues of hirudin in the formation of its complex with a-thrombin. The contribution to binding energy of acidic residues in the 11 Cterminal amino acids varied from 2.3 to 5.9 kJ mol-1. The differences between the contributions of individual residues were smaller than would be expected from the crystal structures of the thrombin-hirudin complex. In particular, the small effect (2.4 kJ mol-1) for the replacement of Asp-55 was surprising in view of the two salt bridges made by this residue.The results of studies involving multiple mutations indicated that the additivity of the effects varied with the position of the mutation. Whereas the effect of mutations involving the glutamic acid residues at positions 61 and 62 were additive, non-additivity was observed with the glutamic acid residues at positions 57 and 58.

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Section: Resultsmentioning

confidence: 99%

Ionic interactions in the formation of the thrombin-hirudin complex

Betz

Hofsteenge

Stone

1991

Biochemical Journal

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“…However, the rms deviation for the 146-150-loop, in which Lys 149E is located, is 4.9 A. The susceptibility of this loop to protease cleavage suggests that it is quite flexible (Berliner, 1984;Brezniak et al, 1990), and crystallography studies suggest that it is held in one conformation only by crystal-packing restraints SkrzypczakJankun et al, 1991). Thus, the conformation of this loop in the crystal structure of the thrombin-rhir complex probably does not represent its structure either in unbound thrombin or in the complex in solution.…”

Section: Discussionmentioning

confidence: 99%

Electrostatic interactions in the association of proteins: An analysis of the thrombin–hirudin complex

et al. 1992

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The role of electrostatic interactions in stabilization of the thrombin-hirudin complex has been investigated by means of two macroscopic approaches: the modified Tanford-Kirkwood model and the finite-difference method for numerical solution of the Poisson-Boltzmann equations. The electrostatic potentials around the thrombin and hirudin molecules were asymmetric and complementary, and it is suggested that these fields influence the initial orientation in the process of the complex formation. The change of the electrostatic binding energy due to mutation of acidic residues in hirudin has been calculated and compared with experimentally determined changes in binding energy. In general, the change in electrostatic binding energy for a particular mutation calculated by the modified Tanford-Kirkwood approach agreed well with the experimentally observed change. The finitedifference approach tended to overestimate changes in binding energy when the mutated residues were involved in short-range electrostatic interactions. Decreases in binding energy caused by mutations of amino acids that do not make any direct ionic interactions (e.g., Glu 61 and Glu 62 of hirudin) can be explained in terms of the interaction of these charges with the positive electrostatic potential of thrombin. Differences between the calculated and observed changes in binding energy are discussed in terms of the crystal structure of the thrombin-hirudin complex.Keywords: electrostatic interactions; hirudin; protein-protein interactions; thrombin Thrombin is a serine protease that plays a central role in blood coagulation. It cleaves fibrinogen to yield fibrin monomers that form the basis of the blood clot. In addition, thrombin activates a number of other proteins involved in coagulation. Thrombin can be distinguished from other serine proteases, such as trypsin, in that it achieves its specificity by using binding sites, called exosites, that are distant from the catalytic center (Fenton, 1988). The crystal structures of thrombin (Bode et al., 1989(Bode et al., , 1992 and its complexes with the polypeptide inhibitor hirudin (Kinemage 1; Griitter et al., 1990;Rydel et al., 1990Rydel et al., , 1991 cleft of thrombin, the C-terminal region binds to a positively charged surface groove called the fibrinogen-recognition exosite. The C-terminal region of recombinant hirudin (rhir) between residues 55 and 65 contains five negatively charged residues, and results from studies using site-directed mutagenesis indicate that each of these negatively charged residues (Asp 55: Glu 57: Glu 58: Glu 61', and Glu 62') contributes to the stabilization of the complex (Braun et al., 1988a;Betz et al., 1991). In addition, the effect of ionic strength on the interaction between thrombin and hirudin suggests that electrostatic interactions with the C-terminal region of hirudin are important for the rate of complex formation and the stability of the complex (Stone et al., 1989). However, although protein engineering studies indicate that each of the negatively char...

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“…Bovine -thrombin was prepared by treatment of ␣-thrombin (27 M, 2 Nomenclature of Schechter and Berger (1). 3 The residues of ␣-thrombin have been numbered after the corresponding amino acids in chymotrypsin using the convention of Bode et al (2) (32). On this basis, the resulting inactivated preparation was designated -IIa i .…”

Section: Preparation Of Thrombin Derivativesmentioning

confidence: 99%

“…3A) yields two peptides that remain noncovalently associated (32,52). The cleavage site separates thrombin into approximate hemispheres, each bearing elements of the catalytic triad, that can be reassociated following dissociation and separation to reconstitute enzymatic activity (52).…”

Section: Dissection Of Interaction Sites Of Thrombin and Prethrombin mentioning

confidence: 99%

Regions Remote from the Site of Cleavage Determine Macromolecular Substrate Recognition by the Prothrombinase Complex

Betz¹,

Krishnaswamy

1998

Journal of Biological Chemistry

116

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The proteolytic formation of thrombin is catalyzed by the prothrombinase complex of blood coagulation. The kinetics of prethrombin 2 cleavage was studied to delineate macromolecular substrate structures necessary for recognition at the exosite(s) of prothrombinase. The product, ␣-thrombin, was a linear competitive inhibitor of prethrombin 2 activation without significantly inhibiting peptidyl substrate cleavage by prothrombinase. Prethrombin 2 and ␣-thrombin compete for binding to the exosite without restricting access to the active site of factor Xa within prothrombinase. Inhibition by ␣-thrombin was not altered by saturating concentrations of low molecular weight heparin. Furthermore, proteolytic removal of the fibrinogen recognition site in ␣-thrombin only had a modest effect on its inhibitory properties. Both ␣-thrombin and prethrombin 2 were cleaved with chymotrypsin at Trp 148 and separated into component domains. The C-terminal-derived 2 fragment retained the ability to selectively inhibit macromolecular substrate cleavage by prothrombinase, while the 1 fragment was without effect. As the 2 fragment lacks the fibrinogen recognition site, the P1-P3 residues or the intact cleavage site, specific recognition of the macromolecular substrate by the exosite in prothrombinase is achieved through substrate regions, distinct from the fibrinogen recognition or heparin-binding sites, and spatially removed from structures surrounding the scissile bond.

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Human .alpha.- to .zeta.-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained

Cited by 56 publications

References 31 publications

Ionic interactions in the formation of the thrombin-hirudin complex

Ionic interactions in the formation of the thrombin-hirudin complex

Electrostatic interactions in the association of proteins: An analysis of the thrombin–hirudin complex

Regions Remote from the Site of Cleavage Determine Macromolecular Substrate Recognition by the Prothrombinase Complex

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