Open AccessDetermination of the Stability Constants of Mn 2+ and Mg 2+ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance
Author(s) -
KUCHEL Philip W.,
REYNOLDS C. Hugh,
DALZIEL Keith
Publication year - 1980
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1980.tb04888.x
Subject(s) - isocitrate dehydrogenase , chemistry , ionic strength , reaction rate constant , ligand (biochemistry) , idh1 , dehydrogenase , electron paramagnetic resonance , crystallography , enzyme , kinetics , nuclear magnetic resonance , biochemistry , receptor , physics , quantum mechanics , aqueous solution , mutation , gene
1. The stability constants ( K s ) of Mn 2+ and Mg 2+ complexes of isocitrate, 2‐oxoglutarate, NADP and NADPH have been estimated by using electron spin resonance to measure free Mn 2+ in ligand–metal‐ion solutions. 2. The values of K s for the Mn 2+ 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 Mg 2+ complexes under the same conditions, the K s 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 Mn 2+ , and the stability constant of the complex is about 2.2 × 10 4 M −1 . The formation of this complex may explain the inhibition of the enzyme‐catalysed reaction observed with Mn 2+ concentrations greater than 0.2 mM in initial rate measurements.