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

Transformation of prophospholipase A into phospholipase A is triggered by the tryptic hydrolysis of the Arg,-Ma, bond of the zymogen. This bond is a better substrate for trypsin than the corresponding strategic bonds of other zymogens such as trypsinogen and chymotrypsinogen A or B: K , = 2.2 mM, k,,t = 7 s-l a t pH 8, 1 "C. Calcium ions which have an essential role in phospholipase catalysis play no role in prophospholipase activation.A comparative physico-chemical analysis of the zymogen and of the enzyme shows that activation produces only very limited changes in the overall folding of prophospholipase. A structural rearrangement occurs at the N-terminal end of the precursor. An excellent fluorescence reporter group to follow this conformational modification is the side-chain of tryptophan-3 (in phospholipase sequence) which passes from a polar to an apolar environment during activation.The a-amino group of alanine-1 (in the phospholipase sequence) which appears on activation is essential for enzyme activity. Selective chemical modification of this function by a number of reagents abolishes phospholipase activity.By analogy with the chymotrypsinogen-chymotrypsin and the proelastase-elastase transformations, it is proposed that the a-amino group is involved in the formation of a salt-bridge which stabilizes the adequate geometry of the active site. Evidences in favor of this hypothesis include the high p K and the low chemical reactivity of the a-amino group, together with the resistance of native phospholipase to aminopeptidase degradation.Phospholipase A is an enzyme catalysing the specific hydrolysis of 3-8%-phosphoglycerides [ 11.This enzyme has been isolated from a number of venoms (see [2a,b]) and from mammalian pancreas Abita, unpublished observations), it has been shown that the pancreatic enzyme is synthesized and secreted exclusively in the form of an inactive precursor, prophospholipase A, which, as other pancreatic enzymes precursors, is activated by trypsin in the duodenum. Complete sequences are known only for phospholipases from porcine pancreas [12,13] and bec venom [14].The porcine enzyme is a single-chain protein consisting of 123 amino acids having a molecular weight of 13 870. N-terminal and C-terminal residues are alanine and half-cystine, respectively. The zymogen is also a single-chain protein of 130 amino acids having a molecular weight of 14610 and an [3-71. N-terminal pyroglutamic acid. The transformation of the zymogen into the active enzyme by tryptic hydrolysis is due to splitting of the Arg,-Ala, bond. It liberates the N-terminal heptapeptide from the precursor, < Clu-Glu-Gly-Ile-Ser-Ser-Arg [15] (where

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