Further studies on the activation of bovine pancreatic procarboxypeptidase A by trypsin

Abstract

Unlike the pancreatic endopeptidase zymogens, procarboxypeptidase A is activated very slowly in vitro. The activation proceeds through the removal of about 100 amino acids away from the N-terminus of the chain. The cleavage of the susceptible bond(s) in monomeric and aggregated forms of bovine procarboxypeptidase A by catalytic amounts of trypsin was found to be very fast. However, as in the case of the porcine zymogen, the expression of the carboxypeptidase activity was considerably delayed by the inhibitory effect of the activation peptide which remains bound to the enzyme molecule after the trypsin treatment of the zymogen. a-Carboxypeptidase A was mainly formed under the relatively mild conditions used, indicating that the Arg-' -Ala" bond is probably the first to be cleaved during in vitro activation.The bovine carboxypeptidase activity was immediately and reversibly expressed upon dimethylmaleylation of the activation mixtures. This expression does not require full dissociation of the enzyme-peptide complex but merely a suitable change in its quaternary structure resulting from a modification of some electrostatic interactions upon dimethylmaleylation.Separation of bovine carboxypeptidase A from its activation peptide was only achieved upon filtration of the dimethylmaleylated mixtures in the presence of 6 M urea. The bovine activation peptide contains at least 93 amino acids compared to the 94 amino acids found by other authors for the rat and porcine peptides and sequencing of the first 53 amino acids showed a 75 -85% homology with the latter two peptides.Pancreatic carboxypeptidase A (CPA) is a zinc exopeptidase that sequentially degrades proteins and peptides by cleavage of certain C-terminal bonds. Its chemical properties and mechanism of action have been extensively studied (for recent reviews see [l, 21). The enzyme is synthesized in the form of the inactive zymogen procarboxypeptidase A (pro-CPA) which, depending on the species, is present in pancreatic juice either as a monomer [3 -61 or an aggregate with one [7, 81 or two [6, 9 -111 other proteins. A ternary complex with a sedimentation coefficient of 6s in which pro-CPA is associated with a chymotrypsinogen of the C-type [I21 and an inactive protein (subunit 111) related to an elastase [13, 141 has so far been described only in three ruminant species: ox, goat and sheep [6]. In the pig, pro-CPA is partly monomeric and We wish to dedicate this paper to the memory of Professor Pierre Desnuelle (Centre National de la Recherche Scientifique, Marseille) for his kind interest during the course of this work and for his helpful criticisms during the preparation of this manuscript.Abbreviations. CPA, carboxypeptidase A; pro-CPA, procarboxypeptidase A.Enzynies. Carboxypeptidase A (EC 3.4.17.1); trypsin (EC 3.4.21.4; chymotrypsin (EC 3.4.21 .1).Note. The numbering system used for amino acid residues corresponds to the following scheme for the conversion of the bovine zymogen into the various forms of CPA porcine CPAs resulting from both tryptic and chym...

Chymotrypsin C Is a Co-activator of Human Pancreatic Procarboxypeptidases A1 and A2

“…Previous studies suggested that chymotrypsin and elastase might activate procarboxypeptidases in the absence of trypsin (16,17,22,24). We found, however, that human elastases and chymotrypsins, other than CTRC, activated proCPA1/ proCPA2 poorly, even after activation with trypsin (see Fig.…”

“…1). Seminal studies on the activation mechanism of bovine, porcine, rat, and human procarboxypeptidases elucidated that activation is initiated by tryptic cleavage at the C terminus of the connecting segment, which destabilizes helix ␣3 and leads to the dissociation of the inhibitory domain (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). In the case of proCPA2 and proCPB, trypsin produced a monophasic activation curve, suggesting that trypsin alone was sufficient to cause propeptide dissociation and complete enzyme activation.…”

“…In porcine proCPA1, these cleavages occurred at a slow rate, and some were assumed to result from atypical trypsin activity at noncanonical sites (19). In addition to the critical role of trypsin, chymotrypsin and elastase were also shown in several studies to catalyze procarboxypeptidase activation to varying degrees; however, neither the mechanism nor cleavage sites involved in these alternative activation pathways have been clarified (16,17,22,24). To date, no convincing attempt has been made to reconcile all biochemical data and define the likely physiological mechanism of procarboxypeptidase activation in the gut.…”

The activation pathway of procarboxypeptidase B from porcine pancreas: Participation of the active enzyme in the proteolytic processing

“…The oligomeric association is the most common occurrence of pro-CPAs (Yamasaki et al, 1963;Uren & Neurath, 1972;Kobayashi et al, 1978;Oppezzo et al, 1994), and it has been observed that the quaternary structure affects their activation behavior (Puigserver & Desnuelle, 1977;Kobayashi et al, 1981; Puigserver et al, 1986;Chapus et al, 1987). No complex with proteinase precursors has yet been described for the B form.…”

Crystal structure of an oligomer of proteolytic zymogens: detailed conformational analysis of the bovine ternary complex and implications for their activation 1 1Edited by I. A. Wilson

“…Interestingly, an enhanced catalytic ef®ciency of activated chymotrypsin C within the complex with bPCPA is observed as compared with the monomeric form (Puigserver & Desnuelle, 1977), maybe due to a more rigid conformation of the active site of this enzyme when bound to bPCPA. It has been further postulated that the slow rise in CPA activity during activation of its zymogen could be due to the inhibitory action of its pro-segment (San Segundo et al, 1982;Chapus et al, 1987;Vendrell et al, 1990). According to this, after tryptic cleavage of the pro-segment the stability of the oligomeric complex would be weakened, given that the activation segment of PCPA acts as a link between the metalloenzyme moiety and PPE, as shown in the porcine system (Vilanova et al, 1985).…”