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 ( K , 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), 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 i-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 effcct on the rate of decomposition of t,his complex. I n 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. 4.The sequence of trypsinogen has been elucidated recently [l, 21 and the covalent changes occurring in the course of the activation of the zymogen have been known for several years [3]. However, there are still two main problems related to the mechanism of the transformation into trypsin. The first one concerns the nature of the morphological changes occurring in the course of the activation which result in the correct positioning of the different elements of the active center. This problem has been studied and is still being studied in this laboratory using both physicochemical and chemical approaches [4-71. The second problem, which is the subject of this paper, is related Unusual Abbreviations. BzArgOEt, a-N-benzoyl-xuginine ethyl ester; AcTyrOEt, N-acetyl-L-tyrosine ethyl ester, to the effect of the unusual sequence Asp-Asp-AspAsp, just preceding the Lys-Ile bond which is hydrolyzed as the first step in the activation process.This sequence has been found in bovine [S, 91, porcine [lo] and ovine [ll] trypsinogens. The peptide sequences liberated in the course of the activation are Val-(Asp),-Lys [S, 9,111 and Phe-Pro-Thr-(Asp),-Lys [10,11]. The kinetic data which relate the effect of a sequence of four aspartyl residues on the rate of hydrolysis of an adjacent trypsin sensitive bond in model peptides, to the mode of activation of bovine and porcine trypsinogens, and of bovine chymotrypsinogens A and B, constitute as far as we are aware, tfhe first detailed study of the proteolytic action of trypsin (and also of aspergillopeptidase A) on a specific bond in large peptides and proteins. Up

Proton magnetic resonance spectra of the cyclic hexapeptides cyclo-(Gly-L-Leu-Gly)2, cyclo-(Gly-cf2-L-Tyr-Gly)2, cyclo-(Gly-L-His-Gly-L-Ala-L-Tyr-Gly), and cyclo(Gly-L-His-Gly-L-Tyr-L-Ala-Gly) were measured, largely at 220 MHz, and compared with the spectra of three previously studied cyclic hexapeptides, cyclo-(Gly-Gly-**4 5oxytocin,6 ferrichrome,6 and evolidine.7 (In each of these there is at least one turn of the peptide chain similar to half the cyclic hexapeptide backbone shown in Figure 1, with a solvent-shielded peptide proton inside that turn.) In the present work we use in addition a recently developed correlation between H-N-C"-H dihedral angle and coupling constant to establish details of conformation. The dihedral angle data are then used in constructing molecular models from which values of the backbone conformational angles and are estimated.

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