Kireeva Eg

The influence of heparin and indol on the catalytic properties of α- and β/δ -thrombins

Abstract Heparin was shown to form an equimolar complex with α- and β/δ -forms of thrombin. The formation of the complex resulted in inhibition of the TAME esterase activity of thrombin ( by 40% form α- and 17% for β/δ-form ) and in stimulation of its BAME esterase activity ( by 50% for α- and 64% for β/δ-form ). Heparin caused the 70% inhibition of the activity of both forms of the enzyme towards the synthetic amid substrate Bz-Phe-Val-Arg-pNA; at the same time it had little if any effect on the enzyme activity towards Tos-Gly-Pro-Arg-pNA and stimulated the α- and β/δ-thrombins activities towards H-D-Phe-Pip-Arg-pNA by 16% and 57% respectively. In the case of both ester and amid substrates heparin exerted its effect on kcat, but had no effect on Km(app). Indol was shown to activate the TAME hydrolysis catalyzed by α- and β/δ-thrombins. The identity of the binding site for indol and for the additional TAME molecule in the effect of substrate activation was demonstrated. Heparin did not prevent the effects of indol and substrate activation of the thrombin-catalyzed hydrolysis of ester substrates. Moreover the kinetic parameters of indol activation are similar for the free enzyme and its complex with heparin indicating the different localization of the binding sites for indol and heparin in the molecule of thrombin.

Substrate activation in the thrombin-catalyzed hydrolysis of synthetic esters of arginine

Abstract The kinetic properties of α-thrombin and product of its limited trypsin-sepharose proteolysis - β-thrombin in wide range of synthetic substrate concentration were studied. Substrate activation was revealed with TAME at substrate concentration higher than 4.0×10−4M. The formation of the intermediate ternary complex is assumed to arise from an interaction of the enzyme with two substrate molecules at high substrate concentration. Kinetic parameters of this complex were calculated. It was shown that the absence of a certain peptide region in α-thrombin molecule (β-form) does not affect the substrate-activating function of thrombin. The increase of KCl concentration from 0.02M to 0.1M activates the enzymatic process. The association of β-thrombin with factor XIII was shown to enhance the esterolytic activity. Estimation of the kinetic constants revealed that the change of activity was due to an increase in the rate of catalysis.