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This is a display of carboxypeptidase A without the substrate. This enzyme is found in the pancreas where it catalyzes the hydrolysis of C-terminal amino acid residues of peptides, proteins and related esters:
PeptideCO-NHCH(R)COOH + H2O PeptideCOOH + NH2CH(R)COOH
It shows a preference for substrates (amino acids) with aromatic side chains such as phenylalanine, tryptophan and tyrosine. Carboxypeptidase has a zinc(II) ion in its active site and was one of the first zinc enzymes to be discovered.
In this display, the alpha-helixes are coloured in blue and the beta-sheets are coloured in red.
The Zn(II) (coloured green) is situated near the protein surface. It has a d10 electronic configuration.
The zinc ion is coordinated to His69 and His196 shown in blue, Glu72 coloured yellow and H2O shown as just the oxygen atom (red).
The active site contains a hydrophobic pocket (red) that promotes the binding of aromatic side chains or in this case the aromatic ring from lactate (light blue).
This is the core of carboxypeptidase A. The three monodentate and a bidentate ligand are arranged in a distorted trigonal bipyramidal fashion about the Zn2+. One glutamate oxygen and nitrogen from His196 occupy the axial, while the other glutamate oxygen, water oxygen and nitrogen on His69 occupy the equatorial positions. Click to see the coordination polyhedron.
The crystal structure of the unproductive complex of carboxypeptidase A formed with an inhibitor, glycyl-L-tyrosine (ball and stick) is displayed. The dotted lines from the amino acid residues to the glycyl-L-tyrosine, symbolize hydrogen bonding. The dotted lines from the inhibitor to the zinc represent electrostatic bonding. This glycine residue chelates zinc using both its amide carbonyl oxygen atom and its terminal amino nitrogen atom. This inhibits the enzyme because the amino group has replaced the water molecule ordinarily coordinated to zinc; therefore the complex is incapable of promoting peptide hydrolysis. The tyrosine ring resides in the hydrophobic pocket, which helps make the enzyme selective for aromatic amino acids.
In the presence of substrates the reaction pathway involves direct attack by water, promoted by Zn2+ and assisted by a Glu 270. The combination of the zinc ion and the neighbouring positively charged residues lower the pKa of the water to approximately 7. A secondary purpose for Zn2+ along with Arg 127, Arg 145 and Tyr 248 is to stabilize the negatively charged intermediates formed during hydrolysis. Here a negatively charged lactate ion (ball and stick) is held in the substrate binding site. This models the amino acid (the leaving group), which is cleaved from the end of the peptide chain where the O of lactate would really be an NH2.
To see a more detailed schematic presentation of the mechanism click here.
This is carboxypeptidase A to explore.
This is a model complex of the active site of carboxypeptidase A. A good model for the carboxypeptidase A active site would be a Zn(II) complex with two N-donor, histidine-like ligands and a carboxylate chelate arranged in such a way to keep one coordination site on Zn2+ centre accessible to other, 2e- donor ligands. The model complex shown on the left contains a in which nitrogen atoms in pyrazolyl rings and carboxyl group mimic the nitrogen atoms from the histidine and COO- group from glutamate residues respectively [1]. The drawback of this ligand is the fact that it can bridge between Zn(II) centres forming a chain structure in the solid state. The existence of two different carbonyl stretching frequencies (at 1668 and 1642 cm-1) in the solution IR spectrum of the complex suggests that the solution contains a mixture of monomeric and polymeric species. Even though this compound resembles the active site of the enzyme, it is not able to assist in peptide bond cleavage.
To learn about simple complexes capable of catalytic hydrolysis of peptide bonds see Chin, Jik (1991) Acc. Chem. Res. 24, 145.