Carboxypeptidase A Zn

Carboxypeplidase A(Co ) not only retains the activity of carboxypeptidase A(Zn ).[Pg.1015]

Carboxypeptidase A is a metalloenzyme—an enzyme that contains a tightly bound metal ion. The metal ion in carboxypeptidase A is Zn. Carboxypeptidase A is one of several hundred enzymes known to contain zinc. In bovine pancreatic carboxypeptidase A, Zn is bound to the enzyme at its active site by forming a complex with Glu 72, His 196, and His 69, as well as with a water molecule (Figure 24.5). (The source of the enzyme is specified because, although carboxypeptidase As from different sources follow the same mechanism, they have slightly different primary stmctures.)... 

The shape of a large protein is influenced by many factors including of course Its primary and secondary structure The disulfide bond shown m Figure 27 18 links Cys 138 of carboxypeptidase A to Cys 161 and contributes to the tertiary structure Car boxypeptidase A contains a Zn " ion which is essential to the catalytic activity of the enzyme and its presence influences the tertiary structure The Zn ion lies near the cen ter of the enzyme where it is coordinated to the imidazole nitrogens of two histidine residues (His 69 His 196) and to the carboxylate side chain of Glu 72... 

FIGURE 27 19 Proposed mechanism of hydrolysis of a peptide catalyzed by carboxypeptidase A The peptide is bound at the active site by an ionic bond between its C terminal ammo acid and the positively charged side chain of arginine 145 Coordination of Zn to oxygen makes the carbon of the carbonyl group more positive and increases the rate of nucleophilic attack by water... 

The body of an adult human contains about 2 g of Zn but, as Zn enzymes are present in most body cells, its concentration is very low and realization of its importance was therefore delayed. The two Zn enzymes which have received most attention are carboxypeptidase A and carbonic anhydrase. 

Peptidyl-dipeptidase A (angiotensin-I converting enzyme, ACE, EC 3.4.15.1) plays a pivotal role in the control of blood pressure [80]. It has been established that its active site contains an essential Zn-atom that functions like that of carboxypeptidase A [2], ACE is inhibited by peptides having a proline or aromatic amino acid at the C-terminal position. These observations as well as the similarities with the active site of carboxypeptidase A have allowed a rational design of effective inhibitors of ACE (e.g., captopril (3.4) and enalapril (3.5)) used in the treatment of hypertension [81]. 

The goal of the experiments we report was to create new structural model complexes for gluzincins or carboxypeptidases. With [Zn (bdtbpza)Cl] (12) for the first time a tetrahedral zinc complex with a monoanionic W,W,0-tridentate using a carboxylate 0-donor was synthesized (41). A comparison of the molecular structure of 12 with the coordination environment of the enzymes indicates its significance... 

Zinc may function to promote the nucleophilicity of a bound solvent molecule in both small-molecule and protein systems. The p/Ca of metal-free H2O is 15.7, and the p/Ca of hexaaquo-zinc, Zn (OH2)6. is about 10 (Woolley, 1975) (Table III). In a novel small-molecule complex the coordination of H2O to a four-coordinate zinc ion reduces the to about 7 (Groves and Olson, 1985) (Fig. 2). This example is particularly noteworthy since it has a zinc-bound solvent molecule sterically constrained to attack a nearby amide carbonyl group as such, it provides a model for the carboxypeptidase A mechanism (see Section IV,B). To be sure, the zinc ligands play an important role in modulating the chemical function of the metal ion in biological systems and their mimics. 

Theoretical studies of carboxypeptidase A suggest that the coordination of the aspartate-histidine couple to zinc, Asp-142-His-69->Zn, facilitates proton transfer from histidine to the carboxylate of aspartate (Nakagawa et al., 1981). Furthermore, Nakagawa etal. (1981) found that... 

Fig. 31. Mechanistic proposal for peptide hydrolysis catalyzed by carboxypeptidase A (Christianson and Lipscomb, 1989). (a) The precatalytic Michaelis complex with substrate carbonyl hydrogen bonded to Arg-127 allows for nucleophilic attack by a water molecule promoted by zinc and assisted by Glu-270 (an outer-sphere C==O Zn interaction is not precluded), (b) Tbe stabilized tetrahedral intermediate collapses, with required proton donation by Glu-270 (Monzingo and Matthews, 1984) Glu-270 may play a bifunctional catalytic role (Schepartz and Breslow, 1987), which results in the product complex (c). [Reprinted with permission from Christianson, D. W., Lipscomb, W. N. (1989) Acc. Chem. Res. 22,62-69. Copyright 1989 American Chemical Society.]...

Fig. 34. Glu-72- Zn interactions in native carboxypeptidase A and in carboxypep-tidase A-inhibitor complexes (inhibitors have been reviewed by Christianson and Lipscomb, 1989). When substrates or inhibitors bind to the enzyme active site and interact with the zinc ion, the interaction of the metal with Glu-72 tends from bidentate toward uniden-tate coordination. The flexibility of protein-zinc coordination may be an important aspect of catalysis in this system, and the Glu-72->Zn - coordination stereochemistry observed here is consistent with the stereochemical analysis of carboxylate-zinc interactions from the Cambridge Structural Database (Carrell et al., 1988 see Fig. 4).

In particular, in Cd(II)-Thermolysin derivative (a zinc metaUoprotease with proteolytic activity similar to carboxypeptidase A), the X-ray structure has provided evidence for isostructural replacement of Zn(II) by Cd(II). In general, the " Cd chemical shift is very sensitive to the nature, number and coordination type of the amino acid ligands and " Cd resonances are commonly detected by direct observation (/ = V2, and 63% sensitivity compared with C) or by inverse detection of Cd scalar-coupled to H. 

Fig. 14. Absorption spectra of Co(II)-carboxypeptidase A, [(CPD)Co], native carboxypeptidase A, [(CPD)Zn], and Co(H20) +, respectively. From Coleman and Valle (94)...

Carboxypeptidase A is a metalloenzyme (containing Zn+ ) which hydrolyzes the C-terminal peptide bond in polypeptide chains (1-4). The hydrolysis occurs most readily when the terminal amino acid residue has an aromatic (or a large aliphatic) R group (cf. 38-> 39+40). 

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...

In carboxypeptidase A [52, 53], the active-site Zn(n) ion plays essential catalytic roles and the guanidinium of Arg-145 recognizes the carboxylate anion of the substrates, thus making the enzyme an exopeptidase. Important features of carboxypeptidase A reproduced by 11 include the essential catalytic action of a metal ion and participation of a guanidinium group in substrate recognition, so that this polymer biocatalyst hydrolyzes unactivated amides, and exhibits selectivity toward amide bonds adjacent to carboxylate groups in the substrate. 

Hydrolysis of peptides and proteins in the GI tract can occur luminally, at the brash border and intracellularly. Luminal activity from the pancreatic proteases trypsin, chymotrypsin, elastase and carboxypeptidase A is mainly directed against large dietary proteins. The main enzymatic activity against small bioactive peptides is derived from the bmsh border of the enterocyte. Brash border proteases, such as aminopeptidase A and N, diaminopeptidease IV and Zn-stable Asp-Lys peptidase, preferentially cleave oligopeptides of up to 10 ammo acid residues and are particularly effective in the cleavage of tri- and tetra-peptides. 

Zn Zn2+ 16 mg Over 200 e.g., carbonic anhydrase, carboxypeptidases Sickle cell anemia causes a Zn deficiency... 

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