Inhibition of carboxypeptidase A by excess zinc: Analysis of the structural determinants by X-ray crystallography

Pancreatic metallocarboxypeptidases are inhibited by a millimolar excess of zinc together with other exo- and endometalloproteases. We have analyzed the structure of bovine carboxypeptidase A inhibited by an excess of zinc ions using Xray crystallography at 1.7 Angstrom overall resolution. Under these conditions, a second zinc is observed to bind to the enzyme active site, establishing a distorted tetrahedrally coordinated complex which involves Glu-270 (the general base for catalysis), a mater molecule, a chloride ion, and a hydroxide ion. This hydroxide ion forms a 114 degrees angular bridge between the inhibitory and the catalytic zinc ions, which are at a distance of 3.3 Angstrom from one another. The inhibitory zinc holds the hydroxide at nearly the same location as a previously observed active site mater molecule (W571) and probably perturbs the substrate positioning and stereochemical rearrangements required for substrate cleavage during catalysis.