Morphology of the procarboxypeptidase A-S6 complex

Bovine pancreatic procarboxypeptidase A is secreted as a non-covalent association of three different proteins (pro CPA-S6). The free native subunits can be obtained by dissociation of the complex by dimethylmaleylation. Moreover, two specific binary complexes resulting from the high affinity of procarboxypeptidase A (subunit I) for its other two partners (subunits II and III) can also be obtained.In order to better understand the function of the association, an investigation of the morphology of the ternary complex by solution X-ray scattering has been carried out. The radii of gyration of all the molecular species have been obtained and the experimental results have been interpreted in terms of compact objects of simple shape. The various components correspond to globular particles as shown by the value of the ratio Rg/M^1/3. This is confirmed by the moderate anisotropy of the simple geometric shapes determined using an assumed value of 0.3 g H₂O/g protein for the hydration. The distances between the centres of gravity of pairs of species strongly suggest that the components are in the closest distance configuration or close to it. However, the binary complex I–III appears to be more open than the complex I–II. Finally, a model of the interaction between carboxpeptidase A and its activation peptide has been constructed by comparing the hypothetical geometric model of subunit I to the crystallographically determined structure of carboxypeptidase A.Abbreviations pro CPA procarboxypeptidase A - pro CPA-S6 (or T.C.) ternary complex with a sedimentation coefficient of 6S - CPA carboxypeptidase A     

The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far.     

Existence of ternary complexes of procarboxypeptidase A in the pancreas of some ruminant species

The existence of procarboxypeptidase A, in the form of a non-covalent ternary complex containing the apparently inactive serine protease (subunit III), has so far been observed only in the ox pancreas. Evidence, obtained in the present study, shows that a ternary complex of procarboxypeptidase A, with a subunit III highly homologous with that of the bovine complex, is also present in two other ruminant species, sheep and goat. The biological significance of these complex forms of procarboxypeptidase A and the consistently high biosynthesis level of the apparently inactive subunit III in all three ruminant species is still unknown. Yet the synthesis of subunit III is not related to the animal diet since in the horse, which is a non-ruminant herbivorous animal, the procarboxypeptidase A is monomeric. Reassociation assays between either bovine subunits II or III and monomeric as well as binary forms of procarboxypeptidase A from various species show that, unlike subunit II, the recognition site for subunit III is highly conserved in all the procarboxypeptidases A and that bovine subunit II is different from porcine chymotrypsinogen C with regard to association.     

Further studies on the human pancreatic binary complexes involving procarboxypeptidase A

In contrast to procarboxypeptidase B which has always been reported to be secreted by the pancreas as a monomer, procarboxypeptidase A occurs as a monomer and/or associated to one or two functionally different proteins, depending on the species. Recent studies showed that, in the human pancreatic secretion, procarboxypeptidase A is mainly secreted as a 44 kDa protein involved in at least three different binary complexes. As previously reported, two of these complexes associated procarboxypeptidase A to either a glycosylated truncated protease E or zymogen E. In this paper, we identified proelastase 2 as the partner of procarboxypeptidase A in the third complex, thus reporting for the first time the occurrence of a proelastase 2/procarboxypeptidase A binary complex in vertebrates. Moreover, from N-terminal sequence analyses, the 44 kDa procarboxypeptidase A involved in these complexes was identified as being of the A1 type. Only one type of procarboxypeptidase B, the B1 type, has been detected in the analyzed pancreatic juices, thus emphasizing the previously observed genetic differences between individuals.     

Comparison between the monomeric and binary-complex forms of procarboxypeptidase A from whole pig pancreas.

Two different forms of procarboxypeptidase A (I and II) were obtained from pig pancreas extracts. The Mr values, the pattern found on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, and the sedimentation coefficients indicate that form I is a binary complex formed by two different subunits, whereas form II is a monomer. The carboxypeptidase A-precursor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx. 12500) and, in the case of the binary complex, the activation with trypsin follows a rather complex pattern, suggesting that the accompanying subunit of form I might play a modulating role in the activation process. Although the appearance of enzymic activity is rather slow, a protein with an Mr equivalent to that of active carboxypeptidase A is found very early in the activation process. Both zymogens are glycoproteins (so far no carbohydrate has been reported in any procarboxypeptidase A) and both contain two strongly bound Zn2+ ions/molecule. Other chemical and physical properties were also determined.     

Identification of a procarboxypeptidase A-truncated protease E binary complex in human pancreatic juice

The characterization, in human pancreatic juice, of a binary complex associating procarboxypeptidase A with a 32 kDa inactive glycoprotein (G32) is reported in this paper. Free G32 was isolated after dissociation of the binary complex. N-terminal sequence analysis revealed a complete homology between this protein and human protease E (HPE 1), except for the two strongly hydrophobic N-terminal residues (Val-Val) which are missing in G32. This protein might be a truncated protease E highly analogous to the subunit III of the ruminant procarboxypeptidase A-S6 ternary complex. The analogy with bovine subunit III is further supported by interspecies reassociation experiments showing that bovine procarboxypeptidase A can specifically bind human G32.     

Primary structure of the activation segment of procarboxypeptidase A from porcine pancreas

The complete primary structure of the activation segment of monomeric procarboxypeptidase A from porcine pancreas has been determined by automated and manual Edman-like degradation methods performed on its fragments generated by enzymatic cleavage. The polypeptide consists of 94 residues, with a molecular mass of 10,768, and presents a high proportion of acidic and hydrophobic residues and a proline-rich region in the center of the molecule. Comparison of this sequence with the already reported equivalent sequence deduced from rat procarboxypeptidase A cDNA reveals a very high degree of homology between the two propeptides (up to a 81% of identities), which is even higher in certain large zones of the molecule.     

Primary structure of the activation peptide from bovine pancreatic procarboxypeptidase A

The complete sequence of the 94 residues composing the activation peptide of bovine procarboxypeptidase A has been determined by automated analysis of the intact activation segment and of three peptides resulting from enzymatic cleavages of the isolated peptide. The sequencing of a CNBr peptide isolated from procarboxypeptidase A allowed to connect the activation peptide with alpha-carboxypeptidase A (peptidylprolyl-L-amino-acid hydrolase, EC 3.4.17.1). The activation segment has a high content of acidic residues and a proline-rich region. Conformational prediction studies show that the bovine peptide, as the porcine and rat peptides, contains a high proportion of secondary structure and that the structural disposition of the regions in secondary structure is similar in the three peptides. The comparison of the sequence of the bovine, porcine and rat peptides, although exhibiting a striking homology, clearly shows that 40% of the substitutions have led to a charge change.     

Reconstitution of bovine procarboxypeptidase A-S6 from the free subunits

The three subunits I, II, and III of bovine procarboxypeptidase A separated by reversible dimethylmaleylation can reassociate to form the reconstituted complexes I + II, I + III, and I + II + III. Since the association II + III is not possible, subunit I appears to play a central role in the formation of the complex. It is suggested that subunit I possesses two independent and specific sites for the recognition of subunits II and III. The liberation of subunit I from any of the complexes was observed to increase its activability, although to a lesser extent than predicted by assays carried out with the succinylated protein. By contrast, the bound form of subunit II was activated faster than the free form. The potential activity of the bound form and the activity of the preformed endopentidase were also higher, suggesting a conformational change induced by association. This suggestion was fully supported by the observed modifications of the heat stability and intrinsic fluorescence spectrum of the subunit resulting form association.     

Intrinsic catalytic activity of procarboxypeptidase A. A kinetic study using fluorine analogues.

Bovine procarboxypeptidase A displays substantial catalytic activity toward halogenated acyl-amino acids, the most active of which is trifluoroacetyl-L-phenylalanine (TFAc-L-Phe). Though this activity is not as great as for the native enzyme, it is quite substantial and far beyond the range of adventitious activation. Both DL-benzylcuccinate and beta-phenylpropionate inhibit zymogen hydrolysis of TFAc-L-Phe, the former with a K1 of 4.1 micrometer and the latter, 900 micrometer (a value much higher than the corresponding enzyme). Apo procarboxypeptidase A will also hydrolyze TFAc-L-Phe, presumably the polarization of the carbonyl carbon being accomplished by the fluorine atoms in the absence of a specific metal ion. That this is not entirely the metal ion function is indicated by the fact that rate enhancements follow the order manganese procarboxypeptidase A approximately zinc procarboxypeptidase greater than apo-procarboxypeptidase. The results indicate considerable similarities for the zymogen-enzyme pair in terms of catalytic groups, pH dependence, specificity and the nature of their transition state binding sites. Some changes in the substrate or inhibitor binding sites are noted.     

Analysis of the activation process of porcine procarboxypeptidase B and determination of the sequence of its activation segment

The molecular events which lead to the proteolytic transformation of porcine procarboxypeptidase B (PCPB) in carboxypeptidase B (CPB) have been determined. Among pancreatic and other tested proteinases, trypsin is the only one capable of generating carboxypeptidase B activity from the zymogen, in vitro. In the first step of this process, trypsin produces cleavage at the boundary between the activation region and the CPB region. Subsequently, a definite sequence of cleavages occurs at the C-terminal end of the released activation segment of 95 residues, giving rise to characteristic intermediates and to a proteolytically resistant activation fragment of 81 residues. In this process, the newly formed CPB participates in the quick-trimming of the released activation peptides. Only a single CPB species is formed in the activation process. This fact and the inability of the released activation peptides to inhibit CPB--and, therefore, their inability to slow down the kinetics of appearance of CPB activity--are two important characteristics differentiating between the activation processes of procarboxypeptidases A and B. The sequence of the 95 residues (MW = 12,835) of the activation region of porcine PCPB has also been deduced, largely from the information obtained by Edman degradation of its fragments and in part by considerations of homology with the rat precursor. The porcine PCPB activation region contains a high percentage of acidic residues, lacks cysteines, methionines, and side-chain posttranslational modifications, and presents a low but significant homology (31%) with the corresponding sequence of porcine procarboxypeptidase A.     

Spectrofluorimetric investigation of the interactions between the subunits of bovine pancreatic procarboxypeptidase A-S6

A spectrofluorimetric investigation of the interactions between the subunits of the pancreatic bovine procarboxypeptidase A ternary complex was carried out after covalent insertion of a fluorescent probe at the active center of one of the constituent subunits. The specific insertion of an anthraniloyl group at the active center of subunit II free or bound to subunit I, after its conversion into chymotrypsin II, allowed us to determine the value of the dissociation constant between subunit I and anthraniloyl-chymotrypsin II (Kd = 0.7 +/- 0.1 x 10(-7) M) and between subunit III and the binary complex subunit I-anthraniloyl-chymotrypsin II (Kd = 1.6 +/- 0.3 x 10(-7) M). Moreover, the influence of the association on the flexibility of the active center of chymotrypsin II was deduced from fluorescence polarization measurements and rotational correlation time determination of anthraniloyl-chymotrypsin II free or bound to subunit I. The anthraniloyl group has no motion independently of the whole chymotrypsin II molecule and the binding of subunit I to anthraniloyl-chymotrypsin II results in an increase of the rigidity of the active site in the latter protein.     

Two-step dissociation of bovine 6S procarboxypeptidase A by dimethylmaleylation

Reversible condensation of the ternary complex form of bovine pancreatic procarboxypeptidase A with 2,3-dimethyl maleic anhydride was investigated at pH 9.0 and low concentration of reagent over the acylable amino groups. After subsequent modification of only a few lysyl residues, subunit III was found to have been released from the quaternary structure leading to the separation of an apparently native protein devoid of any contaminating subunit II, while dissociation of the remaining binary complex occurred upon further addition of the anhydride. This observation suggests that the electrostatic interactions existing between subunits I and III are more rapidly weakened than those between subunits I and II, probably because fewer lysyl residues are involved and/or there is greater accessibility to the chemical reagant. Although completely inactive on the specific substrates of trypsin, chymotrypsin and elastase, subunit III hydrolyzed p-nitrophenyl acetate at a rate similar to that of chymotrypsin but without any burst of p-nitrophenol, which indicates that the weakly functional active site of the subunit is not quite comparable to that of serine protease zymogens. Subunit III already has some of the functional characteristics of the corresponding active enzymes.     

The three-dimensional structure of human procarboxypeptidase A2. Deciphering the basis of the inhibition, activation and intrinsic activity of the zymogen.

The three-dimensional structure of human procarboxypeptidase A2 has been determined using X-ray crystallography at 1.8 A resolution. This is the first detailed structural report of a human pancreatic carboxypeptidase and of its zymogen. Human procarboxypeptidase A2 is formed by a pro-segment of 96 residues, which inhibits the enzyme, and a carboxypeptidase moiety of 305 residues. The pro-enzyme maintains the general fold when compared with other non-human counterparts. The globular part of the pro-segment docks into the enzyme moiety and shields the S2-S4 substrate binding sites, promoting inhibition. Interestingly, important differences are found in the pro-segment which allow the identification of the structural determinants of the diverse activation behaviours of procarboxypeptidases A1, B and A2, particularly of the latter. The benzylsuccinic inhibitor is able to diffuse into the active site of procarboxypeptidase A2 in the crystals. The structure of the zymogen-inhibitor complex has been solved at 2.2 A resolution. The inhibitor enters the active site through a channel formed at the interface between the pro-segment and the enzyme regions and interacts with important elements of the active site. The derived structural features explain the intrinsic activity of A1/A2 pro-enzymes for small substrates.     

Bovine procarboxypeptidase A: kinetics of peptide and ester hydrolysis.

Bovine procarboxypeptidase A exhibits intrinsic hydrolytic activity toward haloacyl amino acids (Behnke and Vallee, 1972), as well as toward conventional peptide and ester substrates for carboxypeptidase A (Bezzone, 1974; Uren and Neurath, 1974). The kinetics of hydrolysis of a series of such substrates by native procarboxypeptidase has now been examined in detail in order to ascertain the extent to which the binding and catalytic sites of carboxypeptidase preexist inthe zymogen. Distinct differences in the substrate binding sites of the zymogen compared with the enzyme are apparent from their respective kinetic profiles as well as from the effects of modifiers on their activities. Substrate activation with the dipeptides BzGly-L-Phe and CbzGly-L-Phe, well known for carboxypeptidase, is exhibited also by the zymogen, but the corresponding substrate inhibition by CbzGly-L-Phe and BzGly-Ophe is absent. Moreover, the substrate inhibition of carboxypeptidase by CbzGlyGly-L-Phe and BzGly-Ophe is replaced by substrate activation in the zymogen...     

Generation of a subunit III-like protein by autolysis of human and porcine proproteinase e in a binary complex with procarboxypeptidase A

Tryptic treatment of human and porcine proproteinase E, procarboxypeptidase A binary complexes gave rise to active proteinase E after removal of an 11-residue N-terminal activation peptide. By contrast, upon treatment of either complex with active proteinase E, not only was the activation peptide released but also the hydrophobic dipeptide Val12-Val13 of the corresponding enzyme. No serine protease activity on specific synthetic peptide substrates could be detected. The structural homology of inactive proteinase E with subunit III of ruminant procarboxypeptidase A was strengthened by the existence of a functional homology since truncated proteinase E still possessed a weakly functional active site. Thus, subunit III-like proteins are generated by proteinase E-catalyzed limited proteolysis of proproteinase E.     

The inactive subunit of ruminant procarboxypeptidase A-S6 complexes. Structural basis of inactivity and physiological role

Subunit III has so far been found only in the pancreas of ruminants in a non-covalent association (procarboxypeptidase A-S6) with two different proteins: the procarboxypeptidase A itself (subunit I) and a C-type chymotrypsinogen (subunit II). In contrast with these latter two proteins, which are zymogens of pancreatic proteases, subunit III seems to be devoid of any activity towards specific substrates of pancreatic proteases. However, it possesses a weakly functional active site which allows it to hydrolyze a non-specific ester, p-nitrophenyl acetate, and to react with several active-site titrants. The binding of proflavin to subunit III shows that this protein owns a non-polar binding site with a very high Kd compared to that of chymotrypsin. The comparison of the amino acid sequences of subunit III and some serine proteases showed that subunit III is closely related to an elastase. Models of the tertiary structure of subunit III suggest a conformational modification that affects the substrate binding and could explain the lack of specific enzymatic activity. The presence of subunit III in the ternary complex is not related to an enzymatic function. This protein does not participate in the activation process of subunit I but prevents the denaturation of this subunit at low pH. This may represent its biological role in the acidic environment of the duodenum in ruminants.     

The tryptic activation pathway of monomeric procarboxypeptidase A

Procarboxypeptidases are the remaining major digestive zymogens the activation process of which remains unsolved. Here it is shown that in the tryptic activation of monomeric procarboxypeptidase A from porcine pancreas, the generation of carboxypeptidase A (CPA) activity parallels the limited proteolysis of the 94-residue activation segment. This degradation proceeds from the COOH-terminal end of the molecule, and CPA itself makes an important and unexpected contribution by excising the COOH-terminal arginine residue of the released primary activation fragment. Successive cleavages at some of the peptide bonds of the activation segment nearest to the COOH terminus were found to be of prime importance in eliciting CPA activity, particularly those involving the carbonyl groups of Arg94 and Gly93 which were first cleaved. It is also shown that the rate of activation does not depend directly upon the generation of CPA-alpha and its conversion to CPA-beta.     

Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity.

Procarboxypeptidase B is converted to enzymatically active carboxypeptidase B by limited proteolysis catalysed by trypsin, removing the long N-terminal activation segment of 95 amino acids. The three-dimensional crystal structure of procarboxypeptidase B from porcine pancreas has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 0.169. The functional determinants of its enzymatic inactivity and of its activation by limited proteolysis have thus been unveiled. The activation segment folds in a globular region with an open sandwich antiparallel-alpha antiparallel-beta topology and in a C terminal alpha-helix which connects it to the enzyme moiety. The globular region (A7-A82) shields the preformed active site, and establishes specific interactions with residues important for substrate recognition. AspA41 forms a salt bridge with Arg145, which in active carboxypeptidase binds the C-terminal carboxyl group of substrate molecules. The connecting region occupies the putative extended substrate binding site. The scissile peptide bond cleaved by trypsin during activation is very exposed. Its cleavage leads to the release of the activation segment and to exposure of the substrate binding site. An open-sandwich folding has been observed in a number of other proteins and protein domains. One of them is the C-terminal fragment of L7/L12, a ribosomal protein from Escherichia coli that displays a topology similar to the activation domain of procarboxypeptidase.