Carboxypeptidase mechanism, hydrogen bonds

Figure 25-19 Steps in a possible mechanism of carboxypeptidase action, (a) The substrate is shown complexed to the enzyme surface through X, Y, Z, and W X is a nonpolar pocket Y is a hydrogen bond, possibly from OH of Tyr 248 Z is the prosthetic group, Zn and W is an...

Bovine carboxypeptidase A was only the third protein, after myoglobin and lysozyme, to have its three-dimensional structure solved at high resolution. The active site zinc is bound to His-69, Glu-72, and His-196 (Figure 12.5), and to a water molecule. The zinc-bound water molecule is itself hydrogen bonded to Glu-270. Despite extensive experimental data, the mechanism of carboxypeptidase still remains controversial. Two major... 

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...

From these studies of the mode of action of carboxypeptidase A, the following two points can be extracted (a) the Zn(II) ion presumably complexes the carbonyl of ester and amide substrates, and (b) Glu-270 is also implicated as a participant, and both general-base and nucleophilic mechanisms have been proposed. There is also strong evidnece that the mechanisms are different with esters and amides. However, another mechanistic possibility to be envisaged for carboxypeptidase A is to consider a nucleophilic attack of the substrate ester or amide bond by Zn(II) coordinated hydroxide ion. Such a possibility was scrutinized by T. H. Fife and V. L. Squillacote (223). In particular, they examined the hydrolysis of the carboxyl substituted ester, 8-quinolyl hydrogen glutarate in the presence of Zn(II) ion. 

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