Mapping the Pro-region of Carboxypeptidase B by Protein Engineering

Abstract

The proteolytic processing of pancreatic procarboxypeptidase B to a mature and functional enzyme is much faster than that of procarboxypeptidase A1. This different behavior has been proposed to depend on specific conformational features at the region that connects the globular domain of the pro-segment to the enzyme and at the contacting surfaces on both moieties. A cDNA coding for porcine procarboxypeptidase B was cloned, sequenced, and expressed at high yield (250 mg/liter) in the methylotrophic yeast Pichia pastoris. To test the previous hypothesis, different mutants of the pro-segment at the putative tryptic targets in its connecting region and at some of the residues contacting the active enzyme were obtained. Moreover, the complete connecting region was replaced by the homologous sequence in procarboxypeptidase A1. The detailed study of the tryptic processing of the mutants shows that limited proteolysis of procarboxypeptidase B is a very specific process, as Arg-95 is the only residue accessible to tryptic attack in the proenzyme. A fast destabilization of the connecting region after the first tryptic cut allows subsequent proteolytic processing and the expression of carboxypeptidase B activity. Although all pancreatic procarboxypeptidases have a preformed active site, only the A forms show intrinsic activity. Mutational substitution of Asp-41 in the globular activation domain, located at the interface with the enzyme moiety, as well as removal of the adjacent 3 10 helix allow the appearance of residual activity in the mutated procarboxypeptidase B, indicating that the interaction of both structural elements with the enzyme moiety prevents the binding of substrates and promotes enzyme inhibition. In addition, the poor heterologous expression of such mutants indicates that the mutated region is important for the folding of the whole proenzyme.

Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors

“…A preliminary purification of B-type carboxypeptidase of H. zea (CPBHz) from the gut content of H. zea larvae allowed its cloning and recombinant expression in the Pichia pastoris system. The cDNA coding for the complete zymogen (PCPBHz) was subcloned into the expression vector pPIC9 and used to transform the KM 71 strain of P. pastoris by using the spheroplasts method as described for other procarboxypeptidases (14,15). The protein was subsequently purified from the supernatant by a first step of hydrophobic interaction chromatography in a butyl-Toyopearl 650M column and a second step of anionexchange on a Sep-Pack Q column, applying a gradient from 100% buffer A (20 mM Tris, pH 8.0) to 100% buffer B (20 mM Tris͞NaCl 1M, pH 8.0) in 30 min.…”

The Structure of Human Prokallikrein 6 Reveals a Novel Activation Mechanism for the Kallikrein Family

“…This construct, harboring three mutations, was used to express the recombinant proteins used in the present study. The protein was heterologously overexpressed in yeast following an approach recently reported for other proproteases (43). Briefly, the vector pPIC9 containing the mutant gene was transformed into Pichia pastoris strain KM71 by the spheroblast method.…”

Characteristics of carboxypeptidase B from pyloric ceca of the starfish Asterina pectinifera

“…CPBs from porcine and bovine pancreases have been isolated and well characterized (Folk et al, 1960;Folk et al, 1962;Wolff et al, 1962;Cox et al, 1962;Wintersberger et al 1962;Schmid & Herriott, 1976;Clauser et al, 1988;Edge et al, 1998;Ventura et al, 1999).…”