The role of divalent cations in the activation of the NADP+-specific isocitrate dehydrogenase from Pisum sativum L.

A divalent cation electrode was used to measure the stability constants (association constants) for the magnesium and manganese complexes of the substrates for the NADP+-specific isocitrate dehydrogenase (EC 1.1.1.42) from pea stems. At an ionic strength of 26.5 mM and at pH 7.4 the stability constants for the Mg2+–isocitrate and Mg2+–NADP+ complexes were 0.85 ± 0.2 and 0.43 ± 0.04 mM−1 respectively and for the Mn2+–isocitrate and Mn2+–NADP+ complexes they were 1.25 ± 0.07 and 0.75 ± 0.09 mM−1 respectively. At the same ionic strength but at pH 6.0 the Mg2+–NADPH and Mn2+–NADPH complexes had stability constants of 0.95 ± 0.23 and 1.79 ± 0.34 mM−1 respectively. Oxalosuccinate and α-ketoglutarate do not form measureable complexes under these conditions. Saturation kinetics of the enzyme with respect to isocitrate and metal ions are consistent with the metal–isocitrate complex being the substrate for the enzyme. NADP+ binds to the enzyme in the free form. Saturation kinetics of NADPH and Mn2+ indicate that the metal–NADPH complex is the substrate in the reverse reaction. In contrast the pig heart enzyme appears to bind free NADPH and Mn2+. A scheme for the reaction mechanism is presented and the difference between the reversibility of the NAD+ and NADP+ enzyme is discussed in relation to the stability of the NADH and NADPH metal complexes.