The linkage between binding of the C-terminal domain of hirudin and amidase activity in human alpha-thrombin

Raimondo De Cristofaro, Bianca Rocca, Raffaele Landolfi, B Bizzi

Risultato della ricerca: Contributo in rivistaArticolo in rivista

Abstract

A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolyl-arginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human alpha-thrombin was developed to dissect the Michaelis-Menten parameters Km and kcat into the individual rate constants of the binding, acylation and deacylation reactions. This method was used to analyse the effect of the C-terminal hirudin (residues 54-65) [hir-(54-65)] domain on the binding and hydrolysis of the three substrates. The results showed that the C-terminal hir-(54-65) fragment affects only the acylation rate, which is increased approx. 1.2-fold for all the substrates. Analysis of the dependence of acylation rate constants on hirudin-fragment concentration, allowed the determination of the equilibrium binding constant of C-terminal hir-(54-65) (Kd approximately 0.7 microM). In addition this peptide was found to competitively inhibit thrombin-fibrinogen interaction with a Ki which is in excellent agreement with the equilibrium constant derived from viscosity experiments. These results demonstrate that binding of hir-(54-65) to the fibrinogen recognition site of thrombin does not affect the equilibrium binding of amide substrates, but induces only a small increase in the acylation rate of the hydrolysis reaction.
Lingua originaleEnglish
pagine (da-a)475-480
Numero di pagine6
RivistaBiochemical Journal
Volume289 ( Pt 2)
Stato di pubblicazionePubblicato - 1993

Keywords

  • Amidohydrolases
  • Amino Acid Sequence
  • Base Sequence
  • Fibrinogen
  • Hirudins
  • Humans
  • Kinetics
  • Mathematics
  • Models, Theoretical
  • Molecular Sequence Data
  • Oligopeptides
  • Peptide Fragments
  • Protein Binding
  • Substrate Specificity
  • Thrombin
  • Viscosity

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