Kinetics and mechanism of catalysis by proteolytic enzymes. The kinetics of hydrolysis of esters of γ-guanidino-l-α-toluene-p-sulphonamidobutyric acid by bovine trypsin and thrombin

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Abstract
Esters of -guanidino-L-[alpha]-toluene-p-sulphonamido-butyric acid ([alpha]-N-toluene-p-sulphonyl-L-norarginine) have been synthesized and shown to be hydrolysed by bovine trypsin and thrombin. As substrates for these enzymes, they were better than esters of [alpha]-N-toluene-p-sulphonyl-L-homoarginine or of [alpha]-N-toluene-p-sulphonyl-L-ornlthine but not as good as esters of [alpha]-N-toluene-p-sulphonyl-L-arginine. With trypsin as catalyst, the methyl and propyl esters are hydrolysed at the same rate at high substrate concentrations and hence deacylation of the acyl-enzyme appears to be rate-determining. In the presence of thrombin, however, the methyl ester is hydrolysed much faster than the [eta]-propyl ester. The variation ofkowith pH indicates that groups with pK(app.) values of 7[middot]05[plus or minus]0[middot]02 and 6[middot]53[plus or minus]0[middot]02 must be dissociated in trypsin and thrombin respectively for hydrolysis to proceed. Activation constrants have been determined for the trypsin-catalysed hydrolysis of methyl [gamma]-guanidino-L-[alpha]- toluene-p-sulphonamidobutyrate and have been compared with the corresponding constants for the hydrolysis of homologous substrates. Cholate increases ko and decreases Km; the effects are more pronounced with thrombin than with trypsin.