COMPARISON OF ESTERASE ACTIVITIES OF TRYPSIN PLASMIN AND THROMBIN ON GUANIDINOBENZOATE ESTERS . TITRATION OF ENZYMES

The acyl-enzyme intermediate, p-guanidinobenzoyl-trypsin, has been shown to deacylate unusually slowly, permitting the use of p-nitrophenyl p′-guanidinobenzoate as a convenient titrant of trypsin. It is now found that thrombin and plasmin, key enzymes in blood clotting physiology which have a trypsin-like esterase specificity, can also be titrated by p-nitrophenyl p′- guanidinobenzoate, showing similar “burst” behavior. This indicates the formation of an acyl-enzyme intermediate in normal plasmin catalyzed reactions, as has been established for the other two enzymes, and strengthens the hypothesis that all three proteolytic enzymes have evolved from a common ancestral form. The kinetic properties of the esterase action of trypsin thrombin, and plasmin on the ethyl and p-nitropheny esters of p′-guanidinobenzoic acid have been characterized. Noteworthy quantitative differences have been found distinguishing thrombin from trypsin and plasmin: the first named is the slowest acylated and the most rapid in deacylation. The esterase action of thrombin appeared even more disparate from the other two proteolytic enzymes in its relative ease of hydrolysis of the isomeric ester, p-nitrophenyl m′-guanidinobenzoate. These findings suggest that the active center of thrombin has greater geometric adaptibility to simple substrates than that of trypsin and plasmin. © 1969, American Chemical Society. All rights reserved.