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

• Published in: European journal of biochemistry Vol. 110; no. 2; pp. 465 - 473
• Main Authors: KUCHEL, Philip W., REYNOLDS, C. Hugh, DALZIEL, Keith
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.09.1980
• Subjects: Drug Stability; Electron Spin Resonance Spectroscopy; Isocitrate Dehydrogenase - metabolism; Ketoglutaric Acids; Kinetics; Magnesium - metabolism; Manganese - metabolism; NADP; Oxidation-Reduction; Protein Binding
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1980.tb04888.x

1. The stability constants (Ks) of Mn2+ and Mg2+ complexes of isocitrate, 2‐oxoglutarate, NADP and NADPH have been estimated by using electron spin resonance to measure free Mn2+ in ligand–metal‐ion solutions. 2. The values of Ks for the Mn2+ complexes at 25°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. 3. 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 thus largely responsible for the observed variation of the apparent equilibrium constant of the NADP‐linked isocitrate dehydrogenase reaction with magnesium ion concentration. 4. NADP‐linked isocitrate dehydrogenase from bovine heart mitochondria binds Mn2+, and the stability constant of the complex is about 2.2 × 104 M−1. The formation of this complex may explain the inhibition of the enzyme‐catalysed reaction observed with Mn2+ concentrations greater than 0.2 mM in initial rate measurements.