Cadmium 113 and carbon 13 NMR studies of ligand binding to pig heart NADP-dependent isocitrate dehydrogenase

Isocitrate dehydrogenase catalyzes the conversion of isocitrate to α-ketoglutarate. The reaction requires a divalent metal. NMR studies using cadmium 113 reveal a resonance in the enzyme-metal-isocitrate complex at 8 ppm relative to Cd(ClO4)2; whereas, in the absence of enzyme, the Cd-isocitrate complex has a resonance at ∼18 ppm. The resonance of enzyme-bound cadmium is typical of cadmium in a complex containing 6 oxygen ligands. Carbon 13 studies were done using specific enrichments at the 1, 2, or 5 positions of α-ketoglutarate and isocitrate, synthesized by enzymatic conversion from glutamate. The carbon 13 resonances of the 1 and 5 carboxyl of α-ketoglutarate are identical in free and enzyme-bound forms over the pH range 5.5-7.5, implying the absence of alterations in geometry of the enzyme-bound form. The 2-carbonyl resonance could not be located in the bound form, suggesting either significant perturbation or immobilization of this group. While the resonances of the carboxyls of free isocitrate shift over the pH range 5-8 reflecting a pK of 5.37, the positions of enzyme-bound resonances remain constant over this pH range. This indicates that isocitrate remains ionized in the enzyme-bound form