Proton NMR spectroscopic characterization of binary and ternary complexes of cobalt(II) carboxypeptidase A with inhibitors

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Abstract
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 .cntdot. I, E .cntdot. I2, and E .cntdot. I .cntdot. 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 .cntdot. I .cntdot. X ternary complexes directly with Co(II)CPD .cntdot. 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. These signals are relatively constant for all E .cntdot. I and E .cntdot. I .cntdot. X complexes, indicating that this ligand is in a relatively fixed or "buried" conformation. However in the 2:1 carboxylate inhibitor (E .cntdot. I2) complexes, both signals are shifted upfield ca. 10 ppm, suggesting a disturbance in the interaction of this histidine with the metal. The other histidine ligand may be more exposed to solvent since its NH is not observed in E, E .cntdot. I, or most E .cntdot. I .cntdot. X and E .cntdot. I2 complexes and, thus, is presumably in rapid exchange. In addition, the C-4 proton signal, d, for this histidine residue varies from 42 to 65 ppm for the binary and ternary complexes, likely reflecting a more labile metal-histidine interaction. In conjunction with crystallographic data, signals a and c are assigned to His-69 and signal d is assigned to His-196.