Search

In order to study the interactions of synthetic substrates with the active site of thrombin the compounds of R-CO-Abz-Arg-OMe were obtained which contained, o-, m- and p-amino benzoic acids (Abz) at P2-subsite. R-CO-moiety was butyric, perfluorobutyric, benzoyl, 2-fluorobenzoyl and perfluorobenzoyl groups. Kinetic parameters of their hydrolysis by thrombin were measured and antithrombin activity was studied. Introduction of fluorine atoms into N-acyl groups caused insignificant alterations of the substrate or inhibitor properties of the compounds containing N-butyric group, but significantly influenced the substrate properties, when peptides contained N-benzoyl group. The interaction mechanism of the obtained substrates with thrombin was proposed.

Hydrolysis and respective catalytic parameters of hydrolysis of ester peptide substrates that contain residues of hydrophobic and nonpolar amino acids in P2, P3 subsites have been studied. It is shown that efficiency of hydrolysis by thrombin is determined by the length of polypeptide chains and by the nature of the amino acids in P2, P3 subsites in the substrate. In spite of the fact that gamma-thrombin retains the conformation activity of the catalytic centre the local conformation changes of the second binding region of the enzyme have been discovered.

A comparative analysis of the temperature effect on the thrombin- and trypsin-catalyzed hydrolysis of synthetic substrates which are derivatives of A(alpha)-chain of fibrinogen has been carried out. The substrates have general formula: Tos-P2-Arg-OCH3 and Tos-P3-P2-Arg-OCH3, where P2-Gly, Val, MeVal; P3 = Gly, Sar. The activation parameters delta G not equal to, delta H not equal to and delta S not equal to are determined. Thrombin is shown to split the most effectively tripeptide containing amino acids sequence Gly-Gly at the subsites of P3 and P2. It is suggested to be caused by an ability of the above compound to take a bent conformation at the active site of thrombin.

Partial dithiothreitol-reduction of the disulphide thrombin bonds when denaturating agents are absent lowers significantly enzymic activity of thrombin relative to fibrinogen coagulation. This permits supposing that at least one of the disulphide bridges in a thrombin molecule is necessary for stabilization of the space structure important for a specific hydrolysis of fibrinogen.

The capacity of methyl esters of arginine-containing substrates to inhibit the proteolytic activity of thrombin is studied. A relationship between the structure of peptides and their capacity to inhibit the thrombin-fibrinogen reaction is investigated. Parameters Ks, k2 and k3, which characterize individual stages of the protein process are calculated from experimentally obtained kcat, Km and I50 values. It is shown that the antithrombin activity of peptides becomes maximal only in the case when residues of hydrophobic amino acids would be simultaneously present both at the positions of P2 and P3 of oligopeptides.