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 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 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 (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., & 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Grissom
C B
CB
Department of Biochemistry, University of Wisconsin, Madison 53706.
Cleland
W W
WW
D000818:Animals; D002247:Carbon Isotopes; D002413:Cations, Divalent; D002645:Chickens; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008433:Mathematics; D008670:Metals / Q000494:pharmacology; D008962:Models, Theoretical; D014783:Viscosity
D002247:Carbon Isotopes; D002413:Cations, Divalent; D008670:Metals; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)