Isotope effect studies of chicken liver NADP malic enzyme: role of the metal ion and viscosity dependence

The role of the metal ion in the oxidative decarboxylation of malate by chicken liver NADP malic enzyme and details of the reaction mechanism have been investigated by 13C isotope effects. With saturating NADP and the indicated metal ion at a total concentration 10-fold higher than its K/sub m/, the following primary 13C kinetic isotope effects at C4 of malate [13(VK/sub mal/)] were observed at pH 8.0: Mg2+, 1.0336; Mn2+, 1.0365; Cd2+, 1.0366; Zn2+, 1.0337; Co2+, 1.0283; Ni2+, 1.025. Knowing the partitioning of the intermediate oxalacetate between decarboxylation to pyuvate and reduction to malate allows calculation of the intrinsic carbon isotope effect for decarboxylation to pyuvate and reduction to malate allows calculation of the intrinsic carbon isotope effect for decarboxylation. For Mg2+ as activator, this was 1.049 with NADP and 1.046 with 3-acetylpyridine adenine dinucleotide phosphate, although the intrinsic primary deuterium isotope effects on dehydrogenation were 5.6 and 4.2, and the partition ratios of the oxalacetate intermediate for decarboxylation as opposed to hydride transfer were 0.11 and 3.96. It was not possible to calculate reasonable intrinsic carbon isotope effects with the other metal ions by use of the partitioning ratio of oxalacetate because of decarboxylation by another mechanism. The variation of 13(VK/sub mal/) with pH was used to dissect the total forward and external components. When the authors attempted to use the variation of 13(VK/sub mal/) with solution viscosity to determine the internal and external commitments, incorrect values were obtained because of a specific effect of the viscosogen in decreasing the K/sub m/ for malate, so that VK/sub mal/ actually increased with viscosity instead of decreasing, as theory predicts