Biochemistry - The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

Types of reactions catalyzed by enzymes
Substitution reactions at carbonyl groups
Involvement of metal ions in the action of proteinases: Carboxypeptidase A

In addition to trypsinogen and chymotrypsinogen, pancreatic juice contains other zymogens that are converted into Enzymes that cleave Amino Acids from the ends of peptide chains (exopeptidases). Unlike Endopeptidases such as Trypsin and Chymotrypsin, they are unable to cleave peptide bonds located within the interior of the polypeptide chain. Carboxypeptidases target exclusively C-terminal groups, sequentially cleaving one amino acid at a time, which makes them a valuable tool for determining the Amino Acid Sequence of Peptides. Carboxypeptidases are also used for protein modification by removing one or more amino acids from the C-terminus. The key feature of Carboxypeptidase A is the presence of a tightly bound zinc ion within its molecule. This ion can be removed from the protein and replaced by other Metal Ions, which in some cases is accompanied by the restoration of enzymatic activity.

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FIG. 7-3. Structure OF THE Active Site of carboxypeptidase A containing a bound peptide substrate (adapted from Lipский [44–46]).

Following the Discovery of the zinc ion in carboxypeptidase, it was immediately hypothesized that it plays a role in catalysis. Unlike protons, which have a weak affinity for the oxygen of the amide carbonyl group, a metal ion can form a strong complex. If THE POSITION OF the metal is fixed by other ligands of the protein molecule, it can be expected that a properly positioned zinc ion will significantly increase the electrophilic character of the carbon atom in the C=O group. Although the three-dimensional structure of carboxypeptidase A has not yet been fully established, X-Ray Diffraction studies of enzyme crystals have confirmed the hypotheses regarding The Role of the zinc ion and provided deeper insight into The Mechanism of the enzymatic reaction [44–46].

The zinc ion forms chelate bonds with two imidazole groups and a glutamic acid side chain (Fig. 7-3). The Arg-245 and Tyr-248 residues form Hydrogen Bonds with the substrate, with the latter acting as a general acid catalyst participating in the protonation of the leaving group. The attacking nucleophile appears to be either the carboxylate group of the Glu-270 residue or a Water molecule. In the former case, an anhydride intermediate must be formed. The presence of a hydrophobic "pocket" explains the enzyme's Specificity toward C-terminal amino acids with bulky hydrophobic side chains.

Another zinc-containing proteinase, Thermolysin produced by Bacillus thermoproteolyticus, is an enzyme characterized by remarkable thermostability. It also contains four bound Calcium Ions [47, 48].