[Determination of the kinetic parameters of individual stages of M(alpha)-arylsulfonyl-L-arginine ester hydrolysis by thrombin and trypsin]

Abstract

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.