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Figure 1.7
Stereo views of the crystal structure of (left) bovine carboxypeptidase A at 1.25 A
(PDB 1ML) with the nucleophilic water shown in red sphere and (right) the enzyme with a
bound potato inhibitor (lavender; PDB 4CPA).
of other proteolytic enzymes such as trypsin, chymotrypsin, and pepsin to aid in the pro-
duction of essential amino acids [43]. Carboxypeptidase A is specific to hydrophobic
C-terminal amino acid residues (such as phenylalanine, tyrosine, or tryptophan), while
the B-type is specific to the charged residues Lys and Arg. Carboxypeptidase A is a mono-
mer of 307 amino acids with a globular shape consisting of both a-helices and b-sheets
(Figure 1.7). The active-site zinc ion plays a key role in the catalysis as it participates in
the stabilization of the intermediate, the deprotonation of the nucleophilic coordinated
water, and the electrostatic interactions critical for recognition of the terminal amino acid
in the substrate peptide chain. This enzyme shares a common active-site motif of
H
69
xxE
72
for zinc binding (with a bidentate carboxylate of E72), along with a second
histidine (H196) located far downstream and a water molecule to complete the coordina-
tion sphere of the metal (Figure 1.7) [44].
As in the case of hexakinase, this protein also undergoes conformational changes upon
substrate binding to the active site “pocket,” which closes upon substrate binding. More
specifically, the negatively charged residues can interact with Arg145 residues in the
active site while the hydrophobic interactions between the substrate and the hydrophobic
pocket can help orient the substrate. Upon binding of potato inhibitor via its C-terminal
carboxylate (Figure 1.7) [45], the bidentate Glu72 residue becomes monodentate which is
accompanied by a
10 A
movement of Tyr248 to form a H-bond with the bound inhibitor
>
2A
movement of the backbone of the region around Tyr248 toward the
metal. Once again, conformational flexibility in metalloproteins is illustrated herein with
carboxypeptidase A during its catalytic action.
along with a
1.2.3.3 Aminopeptidase and Alternative Catalysis
Aminopeptidases are widely distributed hydrolytic enzymes catalyzing various processes,
such as peptide digestion and hormone production, some requiring metal ion(s) in the
active site for full activity. The nuclearity of the active site of metallopeptidases varies
from mononuclear in the case of carboxypeptidase, to dinuclear in aminopeptidases, and
trinuclear in phospholipase C. Even among the dinuclear aminopeptidases, such as those
isolated from bovine lens (bAP) [46],
Escherichia coli
(eAP) [47],
Aeromonas proteoly-
tica
(aAP) [48], and
Streptomyces griseus
(sAP) [49], there is a variation in the structural
and mechanistic roles of the metal ions [50]. For example, the dinuclear aAP shows selec-
tive metal binding, but mononuclear catalytic activity with the second metal playing a
regulatory role [51]. In contrast, the dinuclear sAP (as well as bAP) exhibits dinuclear
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