Maloney R J, Dennis D T
Can J Biochem. 1977 Sep;55(9):928-34. doi: 10.1139/o77-139.
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 alpha-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.
使用二价阳离子电极测量了豌豆茎中NADP⁺特异性异柠檬酸脱氢酶（EC 1.1.1.42）底物的镁和锰配合物的稳定常数（缔合常数）。在离子强度为26.5 mM且pH为7.4的条件下，Mg²⁺-异柠檬酸和Mg²⁺-NADP⁺配合物的稳定常数分别为0.85±0.2和0.43±0.04 mM⁻¹，而Mn²⁺-异柠檬酸和Mn²⁺-NADP⁺配合物的稳定常数分别为1.25±0.07和0.75±0.09 mM⁻¹。在相同离子强度但pH为6.0的条件下，Mg²⁺-NADPH和Mn²⁺-NADPH配合物的稳定常数分别为0.95±0.23和1.79±0.34 mM⁻¹。在这些条件下，草酰琥珀酸和α-酮戊二酸不会形成可测量的配合物。酶对异柠檬酸和金属离子的饱和动力学与金属-异柠檬酸配合物是酶的底物一致。NADP⁺以游离形式与酶结合。NADPH和Mn²⁺的饱和动力学表明，金属-NADPH配合物是逆反应中的底物。相比之下，猪心酶似乎结合游离的NADPH和Mn²⁺。本文提出了反应机制的示意图，并结合NADH和NADPH金属配合物的稳定性讨论了NAD⁺和NADP⁺酶的可逆性差异。
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