Specificity of thrombin: evidence for selectivity in acylation rather than binding for p-nitrophenyl α-amino-p-toluate

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Abstract
The lysyl ester analogue p-nitrophenyl .alpha.-amino-p-toluate hydrobromide was synthesized, and its reactions with human thrombin, trypsin and plasmin were studied by stopped-flow and conventional methods. Kinetic parameters were compared with those determined for the arginyl ester analogue, p-nitrophenyl p-guanidinobenzoate hydrochloride, with these enzymes. By following nitrophenol release or proflavin absorption changes in the stopped-flow spectrophotometer, the constants Ks (enzyme-substrate binding), k2 (acylation), and k3 (deacylation) were determined. Ks values were similar regardless of the substrate or the enzyme and k3 was approximately the same for the reaction of the lysyl ester analogue with any enzyme. K2 for the lysyl ester analogue was 1100 times greater with trypsin than with thrombin and k2 with thrombin was 60 times greater for the arginyl than for the lysyl ester analogue. The limited cleavage of lysyl bonds by thrombin is apparently due in part to restricted acylation rather than substrate binding. The active site of thrombin, compared with that of trypsin, appears to have a more stringent requirement for the spatial relationship between the cationic group and the bond cleaved in substrates.