Carboxypeptidase bovine

Following myoglobin and lysozyme, bovine carboxypeptidase A was the third protein to have its 3-D structure solved at high resolution. The active site zinc is bound to His69, Glu72 and Hisl96 (Figure 12.4), and to a water molecule, which is displaced when a... 

The carboxypeptidases are released from their inactive precursors in the pancreatic juice of animals. The most studied example is bovine carboxypeptidase A, which contains one mole of zinc per protein molecular weight of 34 500. These enzymes cleave the C-terminal amino acid residue from peptides and proteins, when the side-chain of the C-terminal residue is aromatic or branched aliphatic of l configuration. At least the first five residues in the substrate affect the activity of the enzyme. The enzyme also shows esterase activity. Esters and peptides inhibit each other competitively, indicating that the peptidase and esterase sites overlap, even if they are not the same. 

Bovine carboxypeptidase A has a molecular weight of 34,000 and is composed of a single peptide chain. Carboxypeptidase B has similar properties and is probably a homologous protein (90). Both enzymes contain one zinc ion per molecule. Carboxypeptidase A has been exten-... 

Fig. 8 Putative member of carboxypeptidase family from H. pylori HP1075) is aligned with homologous members of bovine carboxypeptidase A. Glu-72, His-169 (Zn binding sites), Arg-145 (carboxylate-binding determinant), and Glu-270 (catalytic residue) are conserved in the H. pylori homologue. However, Zn binding residues corresponding to His-69 are substituted by Gin in the Helicobacter protein sequence...

Bovine carboxypeptidase A is produced in the pancreas as a zymogen, procarboxypeptidase A, MW = 87,000. The proenzyme is composed of three polypeptide chains (151, schematically shown in Figure 15). On limited digestion with trypsin one or more peptide bonds in subunit II is split resulting in its conversion to an enzyme (ATEEase) having activity on acetyl-L-tyrosine ethyl ester similar to that of chymotrypsin. Continued... 

Bovine carboxypeptidase A is likely to be formed by a single chain beginning with an asparagine residue (130) and terminated by the sequence (Glu,Leu,Thr,Val)Asp(NH2) (131). 

Amino Acid Composition of Bovine Carboxypeptidase A and Porcine Carhoxypeplidase B... 

The molecular details of the action of metalloenzymes have begun to be elucidated in the past few years (42). Crystal structures for bovine carboxypeptidase A (43), thermolysin (44), and horse liver alcohol dehydrogenase (45) are now available, and chemical and kinetic studies have defined the role of zinc in substrate binding and catalysis. In fact, many of the significant features elucidating the mode of action of enzymes in general have been defined at the hands of zinc metalloenzymes. 

Interestingly, the successful design of captopril used simple chemical concepts guided by a hypothetical, paper-and-pencil model of substrate and inhibitor binding to the enzyme active site, that had been inferred from the crystal structure of bovine carboxypeptidase A. The X-ray structure of human ACE became available only in 2003, 25 years after the discovery of the captopril class of drugs. While the crystallographic analysis of the ACE complex... 

Figure 5 Multiple sequence alignment of carboxypeptidases. Similarity of the enzymatically active subunit of human carboxypeptidase N (7) to carboxypeptidase A from bovine (1), rat (3), human mast cell (4), bovine carboxypeptidase B (2), human carboxypeptidase M (5), and bovine carboxypeptidase H (6). Residues identical and conservative changes in at least four proteins are boxed. Arrows indicate active site residues. (From N Refs. 119-123.)... 

Bioaffinity chromatography is also a useful tool for the solution of the mechanism of enzymatic processes [140]. Akanuma et al. [141] employed this method for the study of the binding site of bovine carboxypeptidase B on the basis of a complex formation with immobilized substrate analogues of basic and aromatic amino acids. The problem of determining peptides of the active sites of enzymes or antibodies can be solved by the isolation of labelled specific peptides as is shown in section 4.7.5.7. Bioaffinity chromatography was also applied to the study of the molecular structures of human fibroblast and leucocyte interpherones [142]. 

Fig. 2. Three-dimensional structural representations for zinc metall-oproteins. Comparison of the zinc ion-protein bonding interactions for zinc requiring enzymes (A—C) with the zinc-insulin hexamer (D, E). (A) Human carbonic anhydrase C, redrawn from Ref. (47) with permission. (B) Bovine carboxypeptidase Ay, redrawn from Ref. 30) with permission. (C) Bacillus thermoprotedyticus thermolysin, redrawn from Ref. 45) with permission. (D) and (E) Porcine Zn-insulin hexamer, taken from Ref. 48) with permission. The composite electron density maps in (D) and (E) show that each of the two zinc atoms present in the hexamer is within inner sphere bonding distance of three solvent molecules and three histidyl imidazolyl groups in an octahedral array about the metal ion. The position of one of the three equivalently positioned solvent molecules is indicated in (D). The electron density map in (E) shows the relative orientations of the three histidyl residues (His-BlO). (The atomic positions of one of the three equivalent histidyl groups are shown)...

Hass, C.A7.. Neurath. H. Affinity labeling of bovine carboxypeptidase AgLeu by V-bromoacetyl-V-methyl-L-phenylalanine. II. Biochemistry 1971. 10. 3541-3546. 

It is noteworthy that three His, Glu, Asp or Cys residues provide zinc ligands for all known enzyme catalytic zinc sites [ 30], Water is the fourth ligand and histidine is by far the most frequent amino acid among the catalytic site residues. Three histidines are found in human carbonic anhydrases 1 and II, p-lactamase, the DD-carboxypeptidase of Streptomyces albus G, adenosine deaminase and astacin [30]. Two histidines are characteristic of bovine carboxypeptidases A and B, thermolysin and Escherichia coli alkaline phosphatase... 

Affinity Labeling of Bovine Carboxypeptidase Bovine carboxy-peptidase A (2 mg/ml in 2 N NaCl-0.2 M Tris-HCl, pH 7.5) is inactivated by incubation with an equal volume of 4 mM [ Cjbromoacetyl-A -methylphenylalanine at 25°. At suitable intervals samples of 2 ml are removed and the protein and small molecules are separated on a column (1.7 X 15 cm) of Sephadex G-25 equilibrated with 0.25 M NaCl-0.05 M Tris-chloride at pH 7.5. Reaction carried out for 24 hr in the dark yields a modified protein containing 2 moles of inhibitor per mole of carboxypeptidase A. If the reaction is performed in the presence of 0.05 M D-phenylalanine, only one inhibitor molecule is incorporated without loss of activity. 

The presence of two similar hormone binding proteins in bovine neiorophysin recalls the occurrence of isoenzymes. Since the neurophysins were isolated from pituitary posterior lobes pooled from many animals it was possible that individual animals of the same species would produce a single neurophysin. Neurath and his co-workers reported that the two isoenzymes of bovine carboxypeptidase Aa occurred separately and also together in the pancreas of different animals. When a study was made of the neurophysins of 15 individual bovine pituitaries both varieties of protein were found in all. Two intriguing problems remain as to whether neurophysins-I and -II are stored in different neurosecretory granules and the biological roles of the two proteins. 

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