Isotope effect studies of chicken liver NADP malic enzyme: role of the metal ion and viscosity dependence
- 19 April 1988
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 27 (8) , 2927-2934
- https://doi.org/10.1021/bi00408a039
Abstract
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 Km, the following primary 13C kinetic isotope effects at C4 of malate [13(V/Kmal)] 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 nad 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 (the result of the different redox potentials of NADP and the acetylpyridine analogue). The close agreement of the intrinsic 13C isotope effects with each other and with the 13C isotope effect for the Mg2+-catalyzed nonenzymatic decarboxylation of oxalacetate of 1.0489 [Grissom, C.B., and Cleland, W.W. (1986) J. Am. Chem. Soc. 108, 5582] indicates a similarity of transition states for these reactions. 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(V/Kmal) with pH was used to dissect the total forward commitment for hydride transfer into its internal (partition ratios of enzyme intermediates not affected by reaction conditions) and external (partition ratios involving substrate dissociation) components. With NADP and Mg2+, the internal commitent was 0.348 and the external commitment was 0.117. When we attempted to use the variation of 13(V/Kmal) 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 Km for malate, so that V/Kmal actually increased with visocity instead of decreasing, as theory predicts. This approach should prove useful, however, for enzymes where nonspecific effects of viscosity do not occur.This publication has 17 references indexed in Scilit:
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