Binding of ligands to the active site of carboxypeptidase A.
We compare the detailed binding modes of the 39-amino acid inhibitor from potatoes, glycyl-L-tyrosine, the ester analogue CH3OC6H4(CO)CH2CH(CO2(-))C6H5, and indole acetate to the exopeptidase carboxypeptidase A (EC 3.4.17.1). In the potato inhibitor, cleavage of the COOH-terminal glycine-39 leaves a new carboxylate anion of valine-38 having one oxygen on zinc and the other as a receptor of a hydrogen bond from tyrosine-248 of carboxypeptidase. Tyrosine-248 also receives a hydrogen bond from the amide proton of the originally penultimate peptide bond between tyrosine-37 and valine-38. This hydrogen bond suggests product stabilization which is available to peptides and depsipeptides but not to esters lacking an equivalent peptide bond (nonspecific esters). Also, this structure may represent the intermediate binding step for the uncleaved substrate as it moves along the binding subsites. In particular, this may be the binding mode for the substrate after association of the COOH-terminal region of the substrate with the residues at binding subsite S2 (tyrosine-198, phenylalanine-279, and arginine-71) and preceding entry into the catalytic site S1'. These stabilized complexes allow some understanding of the effect of indole acetate, shown here to bind in the pocket at S1', as a competitive inhibitor for esters (for which entry into S1' precedes the rate-determining catalytic step for hydrolysis) and as a noncompetitive inhibitor for peptides (for which entry into S1' is rate limiting). These results, including the binding mode of the ester analogue, are consistent with the original proposal from x-ray studies that both esters and peptides are cleaved with the carboxy terminus at S1', although not necessarily by the same chemical steps.
D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D043422:Carboxypeptidases A; D002384:Catalysis; D003461:Crystallography; D004151:Dipeptides / Q000378:metabolism; D007210:Indoleacetic Acids / Q000494:pharmacology; D008024:Ligands; D010666:Phenylpropionates; D011422:Propionates / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D014443:Tyrosine
D004151:Dipeptides; D007210:Indoleacetic Acids; D008024:Ligands; D010666:Phenylpropionates; D011422:Propionates; D011480:Protease Inhibitors; D014443:Tyrosine; C026788:2-benzyl-3-(4-methoxybenzoyl)propionic acid; D002268:Carboxypeptidases; D043422:Carboxypeptidases A