[Effect of temperature on thrombin- and trypsin-catalyzed hydrolysis of synthetic substrates]. / Vliianie temperatury na kataliziruemyi trombinom i tripsinom gidroliz sinteticheskikh substratov.

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.

[Antithrombin activity of methyl esters of arginine-containing peptides]. / Antitrombinovaia aktivnost' metilovykh éfirov argininsoderzhashchikh peptidov.

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.

[Interaction of an analog of the E1 site of tetrapeptide Gly-Pro-Arg-Pro with the D1 site of two forms of monomeric fibrin]. / Vzaimodeistvie analoga tsentra E1-tetrapeptida Gly-Pro-Arg-Pro s tsentrom D1 dvukh form monomernogo fibrina.

Two monomeric fibrin forms differing in a set of polymerization sites (fibrin desAA and fibrin-desAABB) are inhibited to a different extent by tetrapeptide Gly-Pro-Arg-Pro which simulates a moiety of polymerization site E1. The lesser sensitivity of fibrin-desAABB polymerization to the inhibiting tetrapeptide is due to the presence of active site E2 in it. A shape of the concentration dependence curve of the inhibitory effect of tetrapeptide Gly-Pro-Arg-Pro on the polymerization of both fibrin types is similar to the previously found curve for fibrinogen and its fragments--specific inhibitors of polymerization. Ca2+ intensifies inhibition of fibrin-desAABB polymerization by tetrapeptide Gly-Pro-Arg-Pro twice as much as that of fibrin-desAA evidently due to the peptide blockage of sites D2. An increase of the ionic strength from 0.15 to 0.3 enhances the inhibitory effect of the tetrapeptide on polymerization of two monomeric fibrin forms.

[Structural bases of thrombin specificity. Role of secondary interactions]. / Strukturnye osnovy spetsifichnosti trombina. Rol' vtorichnykh vzaimodeistvii.

The action of thrombin on the esters, Tos-P2-Arg-OCH3 and Tos-P3-P2-Arg-OCH3, where P2 and P3 are the residues of L-, D- or N-methylamino acids, has been studied. The values of kcat and Km(app) under steady-state conditions at pH 8.5 have been determined. The results obtained and literature data suggest that one of the causes of the low catalytic potency of thrombin is a decrease in the hydrophobicity of the environment of Asp-102 located at the active site of the enzyme. Therefore thrombin can cleave only the peptides which hydrophobically shield Asp-102. The key role in this process may be played by the side chains of the amino acids at P2 and P9, thus suggesting the presence of a beta-turn in the P7-P4 region of the polypeptide substrates of thrombin.

[Hydrolysis of methyl esters of N alpha-arylsulfonyl-arginine and N-arylsulfonyl-valyl-arginine by alpha- and beta/gamma-thrombins]. / Gidroliz metilovykh éfirov N. al'fa-arilsul'fonil-arginina i N-arilsul'fonil-valil-arginina al'fa i beta/gamma-trombinami.

The kinetic parameters of hydrolysis of methyl esters of N alpha-arylsulfonyl-arginine and N-arylsulfonyl-valyl-arginine by alpha- and beta/gamma-thrombins were calculated. It was found that the polarity and volume of arylsulfonyl substitute are essential for hydrolysis of N alpha-arylsulfonyl-arginine esters and only slightly affect the hydrolysis of N-arylsulfonyl-valyl-arginine esters. Incorporation of the valine residue into the substrate molecule sharply increases the catalytic constant, thus suggesting an important role of secondary sites of the enzyme binding to the substrate. A comparison of alpha- and beta/gamma-thrombins did not reveal any substantial differences in their ability to hydrolyze synthetic esters; consequently the structure of the region around the enzyme active center which is involved in interactions with the substrates under study, is not responsible for alpha-thrombin specificity for fibrinogen.

[Comparative study of hydrolysis of methyl esters of N-arylsulfonyl-valyl-arginine by thrombin and trypsin]. / Sravnitel'noe issledovanie gidroliza metilovykh éfirov N-arilsul'fonil-valil-arginina trombinom i tripsinom.

The esterase action of thrombin and trypsin on N-arylsulfonyl-valyl-arginine methyl esters was studied. The values of Km and kcat under steady-state conditions at pH 8,5 were determined. It was shown that the nature of the arylsulfonyl group does not affect the kinetic parameters of the reactions under study. The Michaelis constants of the thrombin-catalyzed reactions appeared to be one order of magnitude lower than the Km values of the corresponding TAME analogs.