Kinetics and mechanism of catalysis by proteolytic enzymes. 2. Kinetic studies of thrombin-catalysed reactions and their modification by bile salts and other detergents

Abstract

Three esters of [alpha]-N-benzoyl-L-arginine are hydrolysed at the same rate by bovine throm-bin and it is concluded that deacylation of ([alpha]-N-benzoyl-L-arginly) thrombin is rate-determining. Ionic strength does not influence k3(app.). Four esters of [alpha]-N-toluene-p-sulphonyl-L-arginine are hydrolysed at different rates by thrombin and it is concluded that acylation is partly rate-determining. Increase of ionic strength inhibits the reaction, but probably the acylation step only is affected. Thrombin, unlike trypsin, does not catalyse the hydrolysis of [alpha]-N-toluene -p-sulphonyl-L-homo-arginine methyl ester. [alpha]-N-Toluene-p-sulphonyl-L-lysine methyl ester is an excellent substrate for thrombin, but the ornithine analogue is only slowly hydrolysed and the kinetics are first-order with respect to substrate. The variation of k3(app) with pH for [alpha]-N-benzoyl-L-arginine ethyl ester and [alpha]-N-toluene-p-sulphonyl-L-arginine methyl ester indicates that a group with pK(app.) 6.6 must be dissociated for hydrolysis to proceed. Cholate and glycocholate ions inhibit the hydrolysis of [alpha]-N-benzoyl-L-arginine ethyl ester and accelerate the hydrolysis of [alpha]-N-toluene-p-sulphonyl-L-arginine methyl ester and [alpha]-N-toluene-p-sulphonyl-L-lysine methyl ester. Possible mechanisms are discussed to account for these observations. For the hydrolysis of [alpha]-N-toluene-p-sulphonyl-L-ornithine methyl ester in the presence of cholate, the kinetics are of the Michaelis-Menten type. Except at very low enzyme concentrations, cholate inhibits the clotting of fibrinogen by thrombin. Non-ionic detergents accelerate the esterase activity of thrombin. [alpha]-N-Toluene-p-sulphonyl-L-homoarginine methyl ester is a potent inhibitor of the esterase and clotting activities of thrombin.