Ionization of isocitrate bound to pig heart NADP+-dependent isocitrate dehydrogenase: 13C NMR study of substrate binding

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Abstract
Isocitrate and .alpha.-ketoglutarate have been synthesized with carbon-13 enrichment at specific positions. The 13C NMR spectra of these derivatives were measured as a function of pH. The magnitudes of the changes in chemical shifts with pH for free isocitrate and the magnesium-isocitrate complex suggest that the primary site of ionization is at the .beta.-carboxyl. In the presence of the enzyme NADP+-dependent isocitrate dehydrogenase and the activating metal magnesium, the carbon-13 resonances of all three carboxyls remain constant from pH 5.5 to pH 7.5. thus, the carboxyls remain in the ionized form in the enzyme-isocitrate complex. The .alpha.-hydroxyl carbon resonance could not be located in the enzyme-isocitrate complex, suggesting immobilization of this group. Magnesium produces a 2 ppm downfield shift of the .beta.-carboxyl but does not change the resonances of the .alpha.- and .gamma.-carboxyls. This result is consistent with metal activation of both the dehydrogenation and decarboxylation reactions. The 13C NMR spectrum of .alpha.-ketoglutarate remains unchanged in the presence of isocitrate dehydrogenase, implying the absence of alterations in geometry in the enzyme-bound form. Formation of the quaternary complex with Mg2+ and NADPH leads to loss of the .alpha.-ketoglutarate resonances and the appearance of new resonances characteristic of .alpha.-hydroxyglutarate. In addition, a broad peak ascribed to the enol form of .alpha.-ketoglutarate is observed. The substantial change in the shift of the .beta.-carboxyl of isocitrate and the lack of significant shifts in the other carboxyls of isocitrate or .alpha.-ketoglutarate suggest that interaction of the .beta.-carboxyl with the enzyme contributes to the tighter binding of isocitrate and may be significant for the oxidative decarboxylation function of isocitrate dehydrogenase.

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