Carboxypeptidase proposed catalytic mechanism

Figure 8. Proposed catalytic mechanism for carboxypeptidase A where Glu270 acts as the nucleophile a) nucleophilic attack by carboxylate oxygen of Glu270 on the carbonyl carbon of the substrate b) forming a tetrahedral intermediate c) the intermediate breaks down to give an anhydride species d) hydrolysis of the acylenzyme leads to product formation and regnerates the free enzyme...

Figure 12.9 Proposed catalytic mechanism for carboxypeptidase A. The C-terminal residue, R represent a bulky, hydrophobic side chain. Carboxypeptidase (EC3.3.4.17.-) promotes the polarization of the scissile carbonyl group by hydrogen bonding to Argl27, the activation of water molecule by Zn and its deprotonation by Glu270. The zinc-hydroxide ion attack on the carbonyl carbon forms the tetrahedral oxyanion transition state. The formation of products requires protonation of the amino leaving group presumably by Glu270...

Most exopeptidases are metalloproteases (exceptions e.g. D-amino acid aminopep-tidase, Salmonella methionine aminopeptidase). Aminopeptidases catalyze the hydrolysis of amino acid residues from the N-terminus of peptide substrates with broad substrate specificity. However, carboxypeptidases hydrolyze C-terminal amino acids with varied substrate specificity. Carboxypeptidase A, which prefers large hydrophobic side chain for the C-terminal residue of peptide substrates, has been extensively investigated (Christianson and Lipcomb, 1989) and its catalytic mechanism is illustrated in Figure 12.9. An analogous mechanism has been proposed for the requiring aminopeptidases (Taylor, 1993). 

Similar reaction mechanisms, involving general base and metal ion catalysis, in conjunction with an OH nucleophilic attack, have been proposed for thermolysin (Ref. 12) and carboxypeptidase A (Refs. 12 and 13). Both these enzymes use Zn2+ as their catalytic metal and they also have additional positively charged active site residues (His 231 in thermolysin and... 

The X-ray structures of enzyme and enzyme-inhibitor complexes permit the anhydride intermediate only in the case of carboxypeptidase, not in the case of thermolysin, since in this enzyme the catalytic Glu-143 is too far away from the substrate carbonyl (Lipscomb, 1983). The proposal that carboxypeptidase works via an anhydride intermediate thus requires the supposition that two very similar enzymes work by different mechanisms. 

In models for carboxypeptidase A we showed the intracomplex catalyzed hydrolysis of an ester by a metal ion and a carboxylate ion [106], which are the catalytic groups of carboxypeptidase A. Some mechanistic proposals for the action of carboxypeptidase involve an anhydride intermediate that then hydrolyzes to the product and the regenerated enzyme. Although we later found convincing evidence that the enzyme does not use the anhydride mechanism in cleaving peptides [96-99], it may well use such a mechanism with esters. In a mimic of part of this mechanism we showed [107], but see also Ref. 108, that we could achieve very rapid hydrolysis of an anhydride by bound Zn2+, which is the metal ion in the enzyme. In another model, a carboxylate ion and a phenolic hydroxyl group, which are in the enzyme active site, could cooperatively catalyze the cleavage of an amide by the anhydride mechanism [109]. 

The carboxypeptidase A, mainly isolated from bovine pancreas, is a metalloprotease that hydrolyzes peptide linkages. Particularly, the catalytic cleavage of the peptide in the presence of the enzyme preferably occurs at the C-terminal amide bond with a large hydrophobic amino acid side chain, such as phenylalanine, because the enzyme has a steady binding pocket for an aromatic ring. A proposed mechanism of initial step is shown in Fig. 2 The zinc ion is coordinated by two histidines, one chelating (rj —)glutamate and one water molecule. 

The crystal structures of several complexes of the metallo enzyme, carboxypeptidase A (CPA)(EC 3.4.17.1), have been examined in considerable detail. The structure of the complex with glycyl tryosine (Gly-Tyr) as been refined to 2.0 A resolution and reveals inter alia interactions between the amide carbonyl oxygen and the catalytically essential zinc, and between the amide nitrogen and the hydroxyl of tryosine-248 (Tyr-248)(Fig. 11). The proposed mechanisms for hydrolysis of peptide and ester bonds by CPA have relied heavily on these crystal structures, but a clear distinction between the possible roles of glutamate-270 (Glu-270) in nucleophilic attack either by general base catalysis (Fig. 11 A) or by covalent any hydride formation (Fig. IIB) remains a major unresolved problem. Indeed, it is not yet certain whether esters and amides are hydrolyzed by CPA via identical mechanisms. 

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