title: Cadmium-113 and magnesium-25 NMR study of the divalent metal binding sites of isocitrate dehydrogenases from pig heart.
abstract: The metal activator sites of NAD(+)-dependent and NADP(+)-dependent isocitrate dehydrogenases from pig heart have been probed using 113Cd- and 25Mg-NMR. In the presence of isocitrate and ADP, a broad resonance for cadmium bound to NAD-dependent isocitrate dehydrogenase was observed (-8 ppm) arising from exchange with isocitrate (-20 ppm) and/or ADP (27 ppm) complexes. The Cd shift with ADP suggests interaction of the metal with the nucleotide ring nitrogen. Increasing shifts with excess ADP are indicative of macrochelate formation. 25Mg-NMR demonstrates that, unlike manganese, magnesium has a similar dissociation constant (1.8 mM) from NADP-dependent isocitrate dehydrogenase as from the enzyme-isocitrate complex (1.1 mM). The extrapolated line width of bound magnesium increases from 674 Hz in the binary complex to 10,200 Hz in the ternary complex. The quadrupole coupling constant, calculated from relaxation rates, is larger in the ternary complex, indicative of greater distortion in the magnesium coordination sphere. The line widths of magnesium complexed to NAD-dependent isocitrate dehydrogenase are broader, as expected for the larger octamer. 113Cd- and 25Mg-NMR both show that the metal sites have anisotropic octahedral symmetry. 25Mg relaxation rates yield correlation times corresponding to motions of a domain with motion independent of the enzyme multimers.
authors: Department of Chemistry and Biochemistry, University of Delaware Newark 19716.
authors: R S
authors: RS
authors: Ehrlich
authors: R F
authors: RF
authors: Colman
mesh_terms: D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000737:chemistry*; D007554:Isotopes; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D009206:Myocardium / Q000201:enzymology*; D013552:Swine
chemical_list: D007554:Isotopes; D002104:Cadmium; D007521:Isocitrate Dehydrogenase; D008274:Magnesium