Chapter 4 Carboxypeptidase A

Auld, David S.; Vallee, Bertl .

doi:10.1016/s0167-7306(09)60018-1

Cited by 29 publications

(25 citation statements)

References 149 publications

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“…Thus, kinetic and spectroscopic studies have shown that different small carboxylic acid anions, such as aliphatic carboxylates, D-amino acids or certain dipeptides (e.g. Gly-Tyr) bind to S1' and S1 subsites, and generally behave as 'soft' carboxypeptidase inhibitors [4,49,631. The proposed inhibitory mechanism for these molecules is varied.…”

Section: Inhibitors Of Carboxypeptidasesmentioning

confidence: 99%

Advances in metallo‐procarboxypeptidases

Avilés

Vendrell

Guasch

et al. 1993

European Journal of Biochemistry

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Our knowledge on the structure and functionality of pancreatic carboxypeptidases is rapidly expanding to include that of their zymogen forms. The recent application of fast and mild isolation procedures, together with modern molecular genetic and biochemical-biophysical characterization approaches, has provided a clearer view of the basic structures and functional states in which these zymogens occur, and their evolutionary relationships. The same holds for related metallocarboxypeptidases, either in the pro or active forms, that have been isolated and characterized in non-digestive fluids and tissues, where they probably play an important role in protein and peptide processing. The determination of the three-dimensional structure of the A and B pancreatic zymogens has revealed the molecular determinants of their inactivity and proteolytic activation. The folding of their 95-residue activation segment in a globular N-terminal domain (74-81 residues) and in a connecting region (20-14 residues), and the specific contacts of these pieces with the substrate binding sites of the enzyme, are important factors in zymogen inhibition. On the other hand, the different length of the a-helical connecting region and the stability of its contacts with the enzyme account for the different activation properties of A and B zymogens.The hydrolysis of the peptide bond at the C-terminus of peptides and proteins in living organisms is performed by enzymes belonging either to the metallo-carboxypeptidase family or to the serine-carboxypeptidase family. The latter contain a reactive Ser residue at the active center and other catalytic residues which resemble and have a similar role to those of the Ser-endopeptidases superfamily [I -31. The residues and conformation of the active center of metallocarboxypeptidases are different, having one atom of Zn2 + directly involved in the catalytic mechanism. The present review refers to this latter class of carboxypeptidases and to their precursors.Pancreatic carboxypeptidases, the digestive enzymes involved in the hydrolysis of alimentary proteins from their Cterminal, are the best known enzymes among metallocarboxypeptidases and their occurrence and fundamental properties are very well documented [4 - (EC 3.4.17.10); CPM, membrane-bound carboxypeptidase M (EC 3.4.17.12); CPN, plasma carboxypeptidase N or kininase I (EC 3.4.17.3). specificity between the A form (CPA, with preference for aliphatic C-terminal residues) and the B form (CPB, with preference for basic C-terminal residues), as well as the tertiary structure and mechanism of action of carboxypeptidase A are well known. A number of non-digestive pancreatic-like carboxypeptidases have also been reported in the recent literature, expanding the field of interest in metallo-carboxypeptidases. In contrast to the active enzymes, and for many years, information on the inactive precursor forms of synthesis and storage, procarboxypeptidases A and B (pro-CPA and pro-CPB), has been limited.The lability of these precursors and their occurrence...

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Section: Inhibitors Of Carboxypeptidasesmentioning

confidence: 99%

Advances in metallo‐procarboxypeptidases

Avilés

Vendrell

Guasch

et al. 1993

European Journal of Biochemistry

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“…CPA) is a zinc metalloexopeptidase of 307 residues (MW = 34 472), and a well-studied enzyme (1)(2)(3)(4)(5)(6). This protease preferentially hydrolyzes peptide or ester bonds at the C-terminus of polypeptide substrates with hydrophobic (aromatic or aliphatic) residues.…”

Section: Introductionmentioning

confidence: 99%

“…The three-dimensional structure of CPA, alone and bound to different organic or organopeptidase inhibitors, has been determined by X-ray diffraction at high resolution (1,(7)(8)(9)(10). These studies allowed definition of four of the five previously proposed substrate binding sites from kinetic analyses (S l', S 1, S2, S3, and S4) (2,4,11,12).…”

Section: Introductionmentioning

confidence: 99%

On the water-promoted mechanism of peptide cleavage by carboxypeptidase A. A theoretical study

Álvarez-Santos¹,

González‐Lafont²,

Lluch³

et al. 1994

Can. J. Chem.

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SILVIA ALVAREZ-SANTOS, ANGELS GONZ~FZ-LAFONT, JOSB M. LLUCH, BALDOMERO OLNA, and FRANCE~C X. A m & . Can. J. Chem. 72,2077Chem. 72, (1994.The water-promoted pathway of peptide cleavage by carboxypeptidase A has been studied by semiempirical (AM1) quantum mechanical calculations. A relative1 large model for the CPA-active site elements plus substrate has been designed, using two 3+ .imidazoles and one acetate as the Zn Iigands, acetate as the proton acceptor (simulating , and N-ethylacetamide as the peptide-like substrate. This model, although simpler than the natural one, is one of the largest used for theoretical calculations on CPA catalytic mechanisms. To ensure that this model is able to mimic the natural system, it has been compared with the structure of the ( G I Y )~-L -T~~ + water + CPA complex resulting from several molecular dynamicslenergy minimization simulations.Among the different steps involved in the water-promoted pathway proposed by Lipscomb's group, the attack of the oxygen atom that comes from the activated water molecule to the carbon atom of the peptide bond of the substrate has been found to be the rate-determining step, with a high enthalpy bamer of 37.9 kcal/mol. However, this enthalpy bamer is dramatically decreased when a positive charge, simulating Arg-127, is included near the scissile carbonyl. The reported results seem to favour the occurrence of the mechanism studied and indicate the limitations of using simple elements for the theoretical analysis of enzyme-catalyzed reactions.SUVIA ALVAREZ-SANTOS, ANGELS GONZ~EZ-LAFONT, JOSB M. LLUCH, BALDOMERO OLNA et FRANCE~C X. Avu&. Can. J. Chem. 72,2077 (1994).Utilisant des calculs semiempiriques de micanique quantique (AMl), on a ttudit la voie de clivage des peptides par la carboxypeptidase A (CPA), catalysCe par l'eau. On a mis au point un modble relativement grand des ClCments du site actif de la CPA avec le substrat qui utilise deux imidazoles et un acitate comme ligands du zn2+, un acCtate comme accepteur de proton (simulant le Glu-270) et le N-CthylacCtamide comme substrat semblable i un peptide. Ce modble, mbme s'il est plus simple que le modble naturel, est un des plus grand qui a Ct C utilisC pour des calculs thCoriques sur les mCcanismes catalytiques de la CPA. Afin de s'assurer que ce modble peut imiter le comportement du systbme naturel, on l'a compare avec la structure du complexe ( G~Y )~-L-Tyr + eau + CPA qui rtsulte de plusieurs simulations de dynamique molCculaire et minimisation de l'energie. Parmi les diffkrentes Ctapes impliqutes dans la voie catalysCe par l'eau proposCe par le groupe de Lipscomb, on a trouvC que 1'Ctape determinant la vitesse de la rkaction est l'attaque de l'atome d'oxygbne, provenant de la molCcule d'eau activCe, sur l'atome de carbone de la liaison peptidique du substrat et qu'elle est accompagnCe d'une bambre enthalpique Clevte (37,9 kcal/mol). Toutefois, il se produit une diminution dramatique de cette barribre lorsqu'on introduit une charge positive, ressemblant i celle de

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“…Predictions from the inferred sequence agreed with the results obtained with the natural proteins and supported the accuracy of the cDNA sequence. 2 Comparison of the porcine amino acid sequence with the amino acid sequence of human pro-CPB shows 83.5% identity and 89% similarity between the two proteins.…”

Section: Resultsmentioning

confidence: 96%

“…Pancreatic carboxypeptidases are zinc metalloenzymes that hydrolyze the C-terminal amino acids from alimentary proteins and peptides (1)(2)(3). Two general types of carboxypeptidases can be distinguished: carboxypeptidase A with a preference for apolar C-terminal residues and carboxypeptidase B (CPB), 1 which displays a preference for C-terminal Lys or Arg residues.…”

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confidence: 99%

Mapping the Pro-region of Carboxypeptidase B by Protein Engineering

Ventura¹,

Villegas²,

Sterner³

et al. 1999

Journal of Biological Chemistry

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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.

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Chapter 4 Carboxypeptidase A

Cited by 29 publications

References 149 publications

Advances in metallo‐procarboxypeptidases

Advances in metallo‐procarboxypeptidases

On the water-promoted mechanism of peptide cleavage by carboxypeptidase A. A theoretical study

Mapping the Pro-region of Carboxypeptidase B by Protein Engineering

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