Carboxypeptidase covalent catalysis

Fig. 8-4 The mechanism of covalent catalysis of the hydrolysis of a C-terminal amino acid residue from a peptide by carboxypeptidase A. The reaction is (a) —> (d), and the bold line structure is the peptide substrate. The C-terminal tyrosine side chain of the substrate shown in (a) is denoted by in (ft), (c), and (d).

The covalent catalysis progresses by formation of an impermanent acyl-enzyme intermediate. It and the (3-lactam binding involve the same serine residue of the enzyme s active site. The three-dimensional features of a penicillin-sensitive D-alanyl-carboxypeptidase-transpeptidase and exact binding site of a penicillin and cephalosporin have been established by X-ray crystallographic studies. One of the results of these experiments is to provide physical support for Tipper and Strominger s (1965) original hypothesis that P-lactam antibiotics and the normal substrate bind on the same locus of the transpeptidase. 

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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