Peptide carbonyl-zinc interactions

The peptide carbonyl oxygen interacts with zinc, withdrawing electrons from carbon. Water is displaced from zinc. Glu270 accepts a proton from water - it acts as a general base - and the hydroxyl anion of water acts as a nucleophile to attack the carbonyl carbon of the peptide bond and forms a negatively charged tetrahedral... 

Carboxypeptidase A was the first zinc enzyme to yield a three-dimensional structure to the X-ray crystallographic method, and the structure of an enzyme-pseudosubstrate complex provided a model for a precatalytic zinc-carbonyl interaction (Lipscomb etai, 1968). Comparative studies have been performed between carboxypeptidase A and thermolysin based on the results of X-ray crystallographic experiments (Argosetai, 1978 Kesterand Matthews, 1977 Monzingoand Matthews, 1984 Matthews, 1988 Christianson and Lipscomb, 1988b). Models of peptide-metal interaction have recently been utilized in studies of metal ion participation in hydrolysis (see e.g., Schepartz and Breslow, 1987). In these examples a dipole-ion interaction is achieved by virtue of a chelate interaction involving the labile carbonyl and some other Lewis base (e.g.. 

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.]...

The free a-amino group of the dipeptide interacts with glutamate at position 270 the y-COOH group is involved and a water molecule is located between the amino and carboxyl groups. Interactions which directly involve the catalytic action of the enzyme are as follows (a) the carbonyl of the peptide bond ligands to a zinc atom, which itself is further bound to two histidines (positions 69,196) and a glutamate... 

C-terminal amino acid while the converting enzyme detaches two. Furthermore, it was known that the active site of carboxypeptidase A comprises three important elements for the interaction with the substrate (Fig. 5.25) an electrophilic centre, establishing an ionic bond with a carboxylic function, a site capable of establishing a hydrogen bond with a peptidic C-terminal function, and an atom of zinc, solidly fixed on the enzyme and serving to form a coordinating bond with the carbonyl group of the penultimate (the scissile) peptidic function. 

The X-ray structural studies offer strong evidence in support of a Lewis acid catalytic role for the active site zinc ion in peptide hydrolysis. Since the carbonyl group is no doubt a much weaker dipole than HaO, the initial (ground-state) interaction between the zinc ion and the substrate carbonyl oxygen must be stabilized by the summation of the weak bonding forces between enz5mie and substrate. The result is to displace the transition-state of the enzyme-catalyzed reaction (relative to its hypothetical nonenzymatic cormterpart) along the reaction coordinate toward the enzyme-substrate complex. 

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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