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 X-ray crystallography at 1.7 A 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 water 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 A from one another. The inhibitory zinc holds the hydroxide at nearly the same location as a previously observed active site water molecule (W571) and probably perturbs the substrate positioning and stereochemical rearrangements required for substrate cleavage during catalysis.
Gomez-Ortiz
M
M
Departament de Bioquímica/Institut de Biologia Fonamental, UniversitatAutònoma de Barcelona, Spain.
Gomis-Rüth
F X
FX
Huber
R
R
Avilés
F X
FX
D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D043422:Carboxypeptidases A; D002384:Catalysis; D002417:Cattle; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology*
D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D015032:Zinc