Isotope effect studies of the role of the metal ions in isocitrate dehydrogenase

Abstract

Pig heart NADP+-dependent isocitrate dehydrogenase [EC 1.1.1.42] requires a metal ion for activity. Under optimum conditions (pH 7.5, Mg2+ present), the C isotope effect is k12/k13 = 0.9989 .+-. 0.0004 for the carboxyl C undergoing decarboxylation and the H isotope effects are VmaxH/Vmax2H = 1.09 .+-. 0.04 and (Vmax/Km)H/(Vmax/Km)2H = 0.76 .+-. 0.12 with threo-D,L-[2-2H]isocitric acid. 2H isotope effects measured by the equilibrium perturbation technique under the same conditions are VH/V2H = 1.20 for the forward reaction and 1.02 for the reverse reaction. Under these conditions the rate-determining step in the enzymatic reaction must be product release. Dissociation of isocitrate from the enzyme-isocitrate complex and the enzyme-NADP+ complex must be 2 or more orders of magnitude slower than the chemical steps. The catalytic activity of the enzyme is about 10-fold lower in the presence of Ni2+ than in the presence of Mg2+. The C isotope effect in the presence of Ni2+ at pH 7.5 is k12/k13 = 1.0051 .+-. 0.0012 and the H isotope effects are VmaxH/Vmax2H = 0.98 .+-. 0.07 and (Vmax/Km)H/(Vmax/Km)2H = 1.11 .+-. 0.14. The rate decrease caused by substitution of Ni2+ for Mg2+ must result from the effects of metal on substrate and product binding and dissociation, rather than effects of metal on catalysis. There is also a large metal effect on the rate of the decarboxylation step, consistent with the view that the carbonyl O of the oxalosuccinate intermediate is coordinated to the metal during decarboxylation.