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. 1993 Sep 1;294(Pt 2):563–567. doi: 10.1042/bj2940563

Effect of temperature on the association step in thrombin-fibrinogen interaction.

M Picozzi 1, R De Cristofaro 1
PMCID: PMC1134492  PMID: 8373370

Abstract

Kinetics of fibrinopeptide A release by human alpha-thrombin at low fibrinogen concentration allowed us to measure the specificity constant, i.e. kcat/Km, for the interaction between the enzyme and human fibrinogen. A study of the dependence of the ratio kcat/Km upon the viscosity of the medium revealed that fibrinogen acts as a 'sticky' substrate, or, in other words, as a substrate that dissociates from the Michaelis complex with a rate comparable with that for acylation of the active site. These experiments allowed us also to compute for the first time the second-order rate constant for thrombin-fibrinogen association. A study of the temperature-dependence of the association rate, carried out over the temperature range spanning from 10 degrees C to 37 degrees C (pH 7.50; I0.15) permitted the estimation of the enthalpy and entropy of activation, delta H++ and delta S++, which were found to be equal to 5.69 +/- 0.77 kJ.mol-1 and -80.25 +/- 1.79 kJ.K-1.mol-1 respectively. In addition, the values of Km for thrombin-fibrinogen reaction were measured at different solution viscosities in order to derive the equilibrium dissociation constant, Ks, of this interaction. These experiments showed that the Ks values for thrombin-fibrinogen binding was equal to 1.8 microM at 25 degrees C. Altogether these results indicated that fibrinogen, though interacting with both the catalytic pocket and the fibrinogen recognition site on the thrombin molecule, dissociates from Michaelis complex with a rate comparable with that shown by amide substrates, which interact only with the catalytic site.

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Selected References

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  1. Ascenzi P., Coletta M., Amiconi G., de Cristofaro R., Bolognesi M., Guarneri M., Menegatti E. Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human alpha-, beta- and gamma-thrombin; a kinetic and thermodynamic study. Biochim Biophys Acta. 1988 Sep 21;956(2):156–161. doi: 10.1016/0167-4838(88)90262-2. [DOI] [PubMed] [Google Scholar]
  2. Blanchard J. S. Buffers for enzymes. Methods Enzymol. 1984;104:404–414. doi: 10.1016/s0076-6879(84)04107-0. [DOI] [PubMed] [Google Scholar]
  3. De Cristofaro R., Di Cera E. Modulation of thrombin-fibrinogen interaction by specific ion effects. Biochemistry. 1992 Jan 14;31(1):257–265. doi: 10.1021/bi00116a035. [DOI] [PubMed] [Google Scholar]
  4. De Cristofaro R., Fenton J. W., 2nd, Di Cera E. Modulation of thrombin-hirudin interaction by specific ion effects. J Mol Biol. 1992 Jul 5;226(1):263–269. doi: 10.1016/0022-2836(92)90138-a. [DOI] [PubMed] [Google Scholar]
  5. Di Cera E., De Cristofaro R., Albright D. J., Fenton J. W., 2nd Linkage between proton binding and amidase activity in human alpha-thrombin: effect of ions and temperature. Biochemistry. 1991 Aug 13;30(32):7913–7924. doi: 10.1021/bi00246a007. [DOI] [PubMed] [Google Scholar]
  6. Doolittle R. F. Structural aspects of the fibrinogen to fibrin conversion. Adv Protein Chem. 1973;27:1–109. doi: 10.1016/s0065-3233(08)60446-5. [DOI] [PubMed] [Google Scholar]
  7. Higgins D. L., Lewis S. D., Shafer J. A. Steady state kinetic parameters for the thrombin-catalyzed conversion of human fibrinogen to fibrin. J Biol Chem. 1983 Aug 10;258(15):9276–9282. [PubMed] [Google Scholar]
  8. Hofsteenge J., Taguchi H., Stone S. R. Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors. Biochem J. 1986 Jul 1;237(1):243–251. doi: 10.1042/bj2370243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hopfner K. P., Di Cera E. Energetics of thrombin-fibrinogen interaction. Biochemistry. 1992 Nov 24;31(46):11567–11571. doi: 10.1021/bi00161a040. [DOI] [PubMed] [Google Scholar]
  10. Lewis S. D., Shields P. P., Shafer J. A. Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin. J Biol Chem. 1985 Aug 25;260(18):10192–10199. [PubMed] [Google Scholar]
  11. Martinelli R. A., Scheraga H. A. Steady-state kinetic study of the bovine thrombin-fibrinogen interaction. Biochemistry. 1980 May 27;19(11):2343–2350. doi: 10.1021/bi00552a010. [DOI] [PubMed] [Google Scholar]
  12. Mihalyi E. Clotting of bovine fibrinogen. Kinetic analysis of the release of fibrinopeptides by thrombin and of the calcium uptake upon clotting at high fibrinogen concentrations. Biochemistry. 1988 Feb 9;27(3):976–982. doi: 10.1021/bi00403a021. [DOI] [PubMed] [Google Scholar]
  13. Mihalyi E. Physicochemical studies of bovine fibrinogen. IV. Ultraviolet absorption and its relation to the structure of the molecule. Biochemistry. 1968 Jan;7(1):208–223. doi: 10.1021/bi00841a026. [DOI] [PubMed] [Google Scholar]
  14. Schmitz T., Rothe M., Dodt J. Mechanism of the inhibition of alpha-thrombin by hirudin-derived fragments hirudin(1-47) and hirudin(45-65). Eur J Biochem. 1991 Jan 1;195(1):251–256. doi: 10.1111/j.1432-1033.1991.tb15701.x. [DOI] [PubMed] [Google Scholar]
  15. Stone S. R., Betz A., Hofsteenge J. Mechanistic studies on thrombin catalysis. Biochemistry. 1991 Oct 15;30(41):9841–9848. doi: 10.1021/bi00105a005. [DOI] [PubMed] [Google Scholar]
  16. Stone S. R., Hofsteenge J. Kinetics of the inhibition of thrombin by hirudin. Biochemistry. 1986 Aug 12;25(16):4622–4628. doi: 10.1021/bi00364a025. [DOI] [PubMed] [Google Scholar]
  17. Wells C. M., Di Cera E. Thrombin is a Na(+)-activated enzyme. Biochemistry. 1992 Dec 1;31(47):11721–11730. doi: 10.1021/bi00162a008. [DOI] [PubMed] [Google Scholar]
  18. de Cristofaro R., Rocca B., Bizzi B., Landolfi R. The linkage between binding of the C-terminal domain of hirudin and amidase activity in human alpha-thrombin. Biochem J. 1993 Jan 15;289(Pt 2):475–480. doi: 10.1042/bj2890475. [DOI] [PMC free article] [PubMed] [Google Scholar]

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