The Mechanism of Activation of Trypsinogen. The Role of the Four N-Terminal Aspartyl Residues

Abstract

1 This paper examines in detail the activation of bovine and porcine trypsinogens and of bovine chymotrypsinogens A and B by trypsin and aspergillopeptidase A. Kinetic data have also been obtained (Km and kcat) for the hydrolysis catalyzed by these proteases of several model peptides with sequences related to the N-terminal sequence of bovine trypsinogen. 2 The N-terminal sequence of (aspartyl)4 residues is not necessary for the recognition of the strategic Lys-Ile bond of trypsinogen. 3 We have shown previously that there are two binding sites for Ca2+ on trypsinogen. One of these sites is constituted by the 2 aspartyl residues which are the nearest neighbours of the important Lys-Ile bond. The saturation of the site by Ca2+ improves the formation of the trypsinogen-trypsin complex; Ca2+ has no effect on the rate of decomposition of this complex. 4 The comparison of the kinetic data for the activation of trypsinogens and chymotrypsinogens A and B on one hand and the comparison of kcat and Km of different model peptides, among them the synthetic nonapeptide Val-(Asp)4-Lys-Ile-Val-Gly, on the other hand, implies that the 4 aspartyl residues confer no special reactivity to the Lys-Ile bond but conversely have a very negative effect. For example kcat= 1.6 sec−1 for chymotrypsinogen B but only 2.5 and 5.5 × 10−3 sec−1 for bovine and porcine trypsinogens. The values of Km are similar. For the peptide Val-(Ala)2-Lys-Ile-Val-Gly kcat= 7.0 sec−1 but kcat between 10−4 and 10−2 sec−1 has been found for the peptide Val-(Asp)4-Lys-Ile-Val-Gly. This very negative effect is observed only with trypsin. In the activations by aspergillopeptidase A, trypsinogen is a much better substrate than chymotrypsinogen. The implications of this exceptionally slow hydrolysis of the Lys-Ile bond are discussed. The problem of the formation of inert proteins in particular appears to be closely related to the very poor quality of this bond as a substrate for trypsin. A mechanism is given for the formation of inert proteins; a similar mechanism also explains the degradative autolysis of trypsin.