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Bovine fibrinogen N-terminal fragments were hydrolyzed by native alpha-thrombin and its nonclotting gamma-form with an additional disrupted anion-binding exosite (ABE). The susceptible bond 19-20 of the bovine fibrinogen A alpha-chain was found to be cleaved by alpha-thrombin more effectively than the gamma-form. The difference between the alpha- and gamma-forms disappeared as a result of the substrate site A alpha 37-54 breakdown and was not observed during the hydrolysis of nonspecific macromolecular substrates, such as chymotrypsinogen and insulin B-chain, the latter being hydrolyzed with equal inefficiency by alpha- and gamma-thrombins. The data obtained suggest that sequence 37-54 of the bovine fibrinogen A alpha-chain contains a thrombin recognition site (TRS) which corresponds to the enzyme ABE. Thus, the hypothesis about the existence of two types of high molecular weight thrombin substrates differing by the presence or absence of TRS previously postulated by the authors has been confirmed. The possibility of the catalytic process enhancement due to the ABE-TRS interaction is discussed.

The ability of native alpha- and non-coagulating gamma-thrombin to catalyze the hydrolysis of nonspecific high molecular weight substrates was studied using chymotrypsinogen and the oxidized insulin B-chain as substrates. The effect of thrombin on chymotrypsinogen was estimated by the appearance of caseinolytic activity measured by the increase in the number of terminal NH2-groups in the 2,4,6-trinitrobenzol sulfonic acid reaction. The same reaction was used to study the hydrolysis of insulin by thrombin. It was found that the destruction of the additional center necessary for fibrinogen proteolysis during the alpha-thrombin conversion to the gamma-form did not affect the enzyme ability to hydrolyze nonspecific protein substrates. It was assumed that the low efficiency of non-physiological high molecular weight substrate hydrolysis by thrombin is due to the lack of specific remote interactions in the regulatory site outside the enzyme active center.

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.

It was shown that selective hydrolysis of the disulfide bridge between the A- and B-chains of human thrombin in the absence of denaturating agents decrease its proteolytic (e.g., fibrinogen-binding), esterase and amidase activities. Both chains remain bound by non-covalent interactions. A preparation of partially reduced thrombin was obtained and its kinetic parameters were determined. The experimental results suggest that the S-S bond connecting the A- and B-chains of thrombin is involved in the stabilization of the enzyme active center.

The N(alpha)-arylsulfonyl-L-arginine ethyl- and propyl esters were synthesized and the kinetics of their hydrolysis by thrombin was studied. The values of kcat and Km were shown to depend on the structure of the leaving group and to decrease in the line: OCH3 greater than OC2H5 greater than OC3H7. Using methanol as an additional nucleophile, the kinetic parameters - k2, k3 and Ks - were measured for both thrombin- and trypsin-catalysed reactions. A similarity of two enzymes at the stage of Michaelis complex formation was revealed: the Ks values for both enzymes were practically identical (18.10(-5)M). The differences between thrombin and trypsin were observed at the stages of chemical conversion of substrates and were especially well-pronounced at the stage of acylation. It was shown that the k2 values for thrombin were lower than that for trypsin and the k2/k3 ratio of TAME hydrolysis by trypsin was equal to 21, while that for thrombin was 4.5. This finding is indicative of an essential role of the acylation step in thrombin-catalysed hydrolysis of the esters under study.

For comparative studies on the esterase activities of thrombin and trypsin N(alpha)-arylsulfonyl-L-arginine methyl esters were synthetised containing in aromatic ring substituents of different polar nature, size and hydrophobicity. The kinetics of their hydrolysis by thrombin and trypsin were measured. Values of Km and kcat in steady-state conditions were determined. It was shown, that thrombin-catalysed hydrolysis was more sensitive than that of trypsin to the nature of substituents of arylsulfonyl group and determined by their polar and steric effects. A line correlation between specificity constants (kcat/Km) and sigma and Es of substituents were demonstrated. The difference in reactivity of compounds under investigation is suggested to depend on alterations of stability of hydrogen bond between arylsulfonylamide nitrogen atom of substrate and the active center of the enzyme due to changes in the acidity of the arylsulfonylamide group affected by substituent of the benzene ring.

Trypsin- and thrombin-catalysed hydrolysis of the Nalpha-aryl-sulfonyl-L-arginine methyl esters was studied. Michaelis constants and kcat were determined. The hydrolysis of the substrates by trypsin appears to depend on the polar nature of substitutes. The thrombin- catalysed reaction was believed to be sensitive to steric effects of the introducted groups.

The A- and B-chains have been isolated from the non-covalent complex of human thrombin A- and B-chains, using selective reduction of the interchain disulfide bridge. The B-chain thus isolated (de-A-thrombin) retains its conformation, which is close to the native one and thus differs considerably from the B-chain isolated from the fully reduced enzyme. Nevertheless, the proteolytic (in terms of fibrinogen clotting) and amidase activities of de-A-thrombin are markedly reduced as compared to the native enzyme and the non-covalent complex of A- and B-chains. It is assumed that the A-chain of thrombin is necessary for normal functioning of the active site of thrombin localized in the B-chain.

It was demonstrated that partial reduction of disulfide bonds in thrombin by dithiothreitol in the absence of denaturating agents leads to a decrease of enzymatic activity with respect to fibrinogen coagulation and tosylarginine methyl ester hydrolysis. Polyacrylamide gel electrophoresis and determination of the number of SH-groups liberated in the course of reduction suggest that the observed inactivation is primarily due to the disruption of the S-S-bridge between the A- and B-chains of thrombin.