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

Abstract

The activation process of porcine pancreatic procarboxypeptidase B (pro-CPB) has been studied in detail by a number of complementary methodologies, and a description of the molecular events that lead to the generation of active carboxypeptidase B (CPB) has been deduced. The generated CPB participates in the degradation of its own activation segment by excising C-terminal residues from fragments produced by tryptic proteolysis. The trimming action of CPB is, however, not essential for the release of a fully functional enzyme, in contrast to what was previously reported for porcine procarboxypeptidase A (pro-CPA). In the model presented here, the activation process is solely dependent on the first tryptic cleavage, at the limit between the activation segment and the enzyme region, and the former piece loses all of its inhibitory capacity once severed from the proenzyme. The use of heterologous inhibitors of CPB activity during the study of the tryptic activation process of pro-CPB has been required for the capture of short-lived, otherwise nondetectable, intermediates. This has allowed a complete description of the process and shown that the first proteolytic action of trypsin can also take place on a second target bond. Structural considerations that take into account the three-dimensional structures of the A and B forms of the proenzymes lead us to propose that the differences in conformation at the region that connects the globular activation domain to the enzyme are the main responsible elements for the differences observed in the activation processes of both proenzymes.Keywords: activation domain; activation segment; inhibition mechanism; limited proteolysis; procarboxypeptidase; zymogen Porcine pancreatic secretion contains considerable amounts of the precursor forms of the two major pancreatic carboxypeptidases: AI and B. Procarboxypeptidase A1 (pro-CPA1) is found in the monomeric state and as a member of a binary complex with proproteinase E, whereas procarboxypeptidase B (pro-CPB) is found only in the monomeric state (Aviles et al., 1993). 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. The Reprint requests to: Francesc X. Aviles, Departament de Bioquimica i Biologia Molecular, Unitat de Ciencies and lnstitut de Biologia Fonamental, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain.Abbreviations: BZS, benzylsuccinic acid; CP, carboxypeptidase; PCI, potato carboxypeptidase inhibitor; MMGETP, 2-mercaptomethyl-3-guanidinethylthiopropanoic acid.-~ ~-~-~-presence of the two monomeric zymogens in porcine pancreas makes this system suitable for comparison studies on their activation processes. The activation process of pancr...

Mapping the Pro-region of Carboxypeptidase B by Protein Engineering

“…The recombinant pro-CPB was fully activated when incubated at 0°C and at 400/1 pro-CPB/trypsin (w/w) ratio. The maturation course for recombinant pro-CPB is identical to that previously reported for the natural form isolated from porcine pancreas (16). A scheme of the proteolytic maturation process is presented in Fig.…”

“…The supernatant was loaded onto a hydrophobic interaction butyl column and eluted with a decreasing gradient of ammonium sulfate. The zymogen-containing fractions were selected for their activity against BGA after tryptic activation and re-purified by fast protein liquid chromatography on an anion exchange column (TSK-DEAE) as reported previously (16). The correct processing of the different recombinant proteins was confirmed by both automated Edman degradation analysis of its N-terminal sequence and mass spectrometry.…”

“…3). Previous studies have shown that Arg-93 may also act as a primary tryptic target when carboxypeptidase inhibitors are added to the activation mixture (16).…”

“…At the top of the figure, the sequence around the primary and secondary processing sites is presented to serve as a guide. Modified from Villegas et al (16).…”

Prediction of a new class of RNA recognition motif

“…Folding studies performed in ADA2h showed an extremely fast two-state kinetics [6], suggesting that ADs could assist in the proenzyme folding. Supporting this hypothesis, recombinant expressions of various forms of CPs in the methylotrophic yeast Pichia pastoris have been proved unsuccessful, whereas the proenzyme forms can be produced at high yields.…”