Ionization of isocitrate bound to pig heart NADP+-dependent isocitrate dehydrogenase: 13C NMR study of substrate binding.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002250:Carbon Radioisotopes; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009206:Myocardium / Q000201:enzymology*; D011485:Protein Binding; D013552:Swine
D002250:Carbon Radioisotopes; D007523:Isocitrates; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)