Determination of the Stability Constants of Mn2+ and Mg2+ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance

Abstract

The stability constants (Ks) of Mn2+ and Mg2+ complexes of isocitrate, 2-oxoglutarate, NADP and NADPH were estimated by using ESR to measure free Mn2+ in ligand-metal-ion solutions. The values of Ks for the Mn2+ complexes at 25.degree. C, in triethanolamine buffer containing NaCl, pH 7.0 and ionic strength 0.15 M, are 497 M-1 for isocitrate, 39 M-1 for 2-oxoglutarate, 467 M-1 for NADP and 943 M-1 for NADPH. For the Mg2+ complexes under the same conditions, the Ks values are 357 M-1, 25 M-1, 133 M-1 and 179 M-1, respectively. The large difference between the stabilities of the isocitrate and 2-oxoglutarate complexes is largely responsible for the observed variation of the apparent equilibrium constant of the NADP-linked isocitrate dehydrogenase reaction with Mg ion concentration. NADP-linked isocitrate dehydrogenase from bovine heart mitochondria binds Mn2+, and the stability constant of the complex is about 2.2 .times. 104 M-1. The formation of this complex may explain the inhibition of the enzyme-catalyzed reaction observed with Mn2+ concentrations greater than 0.2 mM in initial rate measurements.