Effect of temperature on the association step in thrombin-fibrinogen interaction.
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.
Centro Ricerche Fisiopatologia dell'Emostasi, Università Cattolica S. Cuore, Roma, Italy.
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M
Picozzi
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R
De Cristofaro
D000215:Acylation; D001665:Binding Sites; D005340:Fibrinogen / Q000378:metabolism*; D005344:Fibrinopeptide A / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013696:Temperature; D013816:Thermodynamics; D013917:Thrombin / Q000378:metabolism*; D014783:Viscosity
D005344:Fibrinopeptide A; D005340:Fibrinogen; D013917:Thrombin