in CPA, and the zinc ion is coordinated by Glu-72, His-69, and His-196 enzyme residues. A plane exists that bisects the active-site groove where residues Glu-270 and Arg-127 are on opposite sides of the zinc-water coupled complex. The zinc is electron rich due to glutamine ligands coordinating the zinc because before substrate binds, Glu-72 coordinates bidentate but shifts to monodentate after substrate binds. As a result, the zinc metal is not able to deprotonate the coordinated water molecule to make a hydroxyl nucleophile. Glu-270 and Arg-127 play an important role in catalysis shown in Figure 2. Arg-127 acts to stabilize the carbonyl of the substrate that is bound to amino group of phenylalanine. Simultaneously, the water molecule coordinated to zinc is deprotonated by Glu-270 and interacts with the carbonyl stabilized by Arg-127. This creates an intermediate, shown in Figure 2, where the negatively charged oxygen is coordinated to zinc, and through unfavorable electrostatic interactions between Glu-270 and the ionized product facilitates the release of the product at the end of catalysis. In recent computational studies, the mechanism of catalysis is similar but the difference in mechanism is that deprotonated water molecule binds to the carbon of the carbonyl, whereas Figure 2 shows the hydroxyl group stays coordinated to zinc. Then proteolysis occurs and the water molecule is then introduced back into the active site to coordinate to zinc. Several studies have been conducted exploring the details of the bond between carboxypeptidase A and substrate and how this affects the rate of hydrolysis. In 1934, it was first discovered through kinetic experiments that, in order for substrate to bind, the peptide that is to be hydrolyzed must be adjacent to a terminal free hydroxyl group. Also, the rate of hydrolysis can be enhanced if the C-terminal residue is branched aliphatic or aromatic. However, if the substrate is a dipeptide with a free amino group, it undergoes hydrolysis slowly; this, however, can be avoided if the amino group is blocked by N-acylation. It is quite clear that the structure of the enzyme, to be specific the active site, is very important in understanding the mechanism of reaction. For this reason, Rees and colleagues studied the enzyme-ligand complex to get a clear answer for the role of the zinc ion. These studies found that, in free enzyme, the zinc coordination number is five; the metal center is coordinated with two imidazole Nδ1 nitrogens,