Carboxypeptidases zinc-carbonyl mechanism

Zinc enzymes catalyze the hydrolysis of amide bonds using a variety of active site structural motifs.77 An extensively studied enzyme of this class is carboxypeptidase A, which contains a mononuclear zinc center (Fig. 13) within the enzyme active site and catalyzes the hydrolysis of a C-terminal amino acid.78,79 The mechanism of amide cleavage in carboxypeptidase A has been studied extensively.77,78 In one proposed mechanistic pathway for this enzyme (Scheme 13), termed the zinc hydroxide mechanism , the zinc center activates a water molecule for deprotonation and also assists in polarization of the substrate carbonyl moiety, thus making it more susceptible to nucleophilic attack. The zinc center also provides transition state stabilization through charge neutralization. 

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. 

One possible mechanism for the hydrolysis of peptides or esters by carboxypeptidase A involves two steps with an anhydride (acyl-enzyme) intermediate.418 In the first step, the zinc(II) activates the substrate carbonyl group towards nucleophilic attack by a glutamate residue, resulting in the production of a mixed anhydride (127). Breakdown of the anhydride intermediate is rate determining with some substrates.419 An understanding of the chemistry of metal ion effects in anhydride hydrolysis is therefore of fundamental importance in regard to the mechanism of action of the enzyme. Until recently there have been few studies of metal ion-catalysed anhydride solvolysis. 

In carboxypeptidase, which cleaves peptide linkages (and also esters), there is first a complex in which the peptide carbonyl oxygen atom coordinates to the zinc ion and then things proceed (at least approximately) as shown in Fig. 15-8. These two mechanisms involve, in somewhat different ways, the electrophilidty of the Zn2+ ion, and it is known or believed that it is this property of the Zn2+ ion, exercised in one way or another, that is critical in all other enzymes having Zn2+ at the active site. 

Figure 9. Proposed catalysic mechanism for carboxypeptidase A where water acts as the nucleophile, (a) nucleophilic attack by a water molecule on the carbonyl carbon of the substrate promoted by zinc and assisted by Glu270 with concommitant transfer of i proton to Glu270 (b) a tetrahedral intermediate, stabilized by interactions with Argl27 and the zinc ion, collapses with a proton donated by Glu270 (c) a second proton transfei results in product formation (d).

The binding of the carbonyl oxygen of amides to zinc(II) of carboxypeptidase is based on X-ray studies of enzyme-inhibitor complexes. Of course, the amides studied may be inhibitors because they bind to the enzyme incorrectly. An alternative mechanism, which makes chemical sense, could involve metal-ion coordination to the amide nitrogen which would not only stabilise the tetrahedral intermediate (XVI) but also facilitate carbon-nitrogen bond cleavage. Such a mode of catalysis... 

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...

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. 

Rapid amide hydrolysis by copper and zinc complexes of a ligand which mimics the carboxypeptidase active site (7) has been observed. The metal is held perpendicular to the amide plane, and at pH 7.6 and 50 °C hydrolysis occurs 2 X 10 times faster in the presence of zinc than in its absence. Copper is a more potent catalyst. A metal-dependent pliTa ( 8.5 for Zn and 7.6 for Cu +) is consistent with a mechanism in which the amide carbonyl is attacked by a co-ordinated hydroxyl nucleophile. A more sophisticated model with a [20]paracylcophane which has a nucleophilic and a metal binding site attached (8) catalyses the hydrolysis of p-nitrophenyl hexadecanoate. The mechanism... 

Based on his study on model compounds, Breslow suggested a second mechanism for peptide hydrolysis by carboxypeptidase A which does not involve the formation of an acyl-enzyme intermediate (221,222). Essentially, in the hydrolysis of a peptide bond there is participation of a zinc ion, a carboxylate ion, and a tyrosine hydroxyl group. The Zn(II) ion still plays the role of a Lewis acid to coordinate the carbonyl oxygen but the carboxylate group rather acts as a general base. The argument is based on the fact that... 

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