1H NMR spectroscopic characterization of binary and ternary complexes of cobalt(II) carboxypeptidase A with inhibitors.
The binding of L- and D-phenylalanine and carboxylate inhibitors to cobalt(II)-substituted carboxypeptidase A, Co(II)CPD (E), in the presence and absence of pseudohalogens (X = N3-, NCO-, and NCS-) has been studied by 1H NMR spectroscopy. This technique monitors the proton signals of histidine residues bound to cobalt(II) and is therefore sensitive to the interactions of inhibitors that perturb the coordination sphere of the metal. Enzyme-inhibitor complexes, E.I, E.I2, and E.I.X, each with characteristic NMR features, have been identified. Thus, for example, L-Phe binds close to the metal ion to form a 1:1 complex, whereas D-Phe binds stepwise, first to a nonmetal site and then to the metal ion to form a 2:1 complex. Both acetate and phenylacetate also form 2:1 adducts stepwise with the enzyme, but beta-phenylpropionate gives a 2:1 complex without any detectable 1:1 intermediate. N3-, NCO-, and NCS- generate E.I.X ternary complexes directly with Co(II)CPD.L-Phe and indirectly with the D-Phe and carboxylate inhibitor 2:1 complexes by displacing the second moiety from its metal binding site. The NMR data suggest that when the carboxylate group of a substrate or inhibitor binds at the active site, a conformational change occurs that allows a second ligand molecule to bind to the metal ion, altering its coordination sphere and thereby attenuating the bidentate behavior of Glu-72. The 1H NMR signals also reflect alterations in the histidine interactions with the metal upon inhibitor binding. Isotropic shifts in the signals for the C-4 (c) and N protons (a) of one of the histidine ligands are readily observed in all of these complexes.(ABSTRACT TRUNCATED AT 250 WORDS)
Bertini
I
I
Department of Chemistry, University of Florence, Italy.
Luchinat
C
C
Messori
L
L
Monnanni
R
R
Auld
D S
DS
Riordan
J F
JF
D000085:Acetates; D019342:Acetic Acid; D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors; D043422:Carboxypeptidases A; D002417:Cattle; D003035:Cobalt; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D010648:Phenylacetates; D010649:Phenylalanine; D010666:Phenylpropionates; D011487:Protein Conformation
D000085:Acetates; D010648:Phenylacetates; D010666:Phenylpropionates; D003035:Cobalt; D010649:Phenylalanine; C035253:3-phenylpropionic acid; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; C025136:phenylacetic acid; D019342:Acetic Acid