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 /sup 13/C 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 /sup 13/C kinetic isotope effects at C/sub 4/ of malate (/sup 13/(VK/sub mal/)) were observed at pH 8.0: Mg/sup 2 +/, 1.0336; Mn/sup 2 +/, 1.0365; Cd/sup 2 +/, 1.0366; Zn/sup 2 +/, 1.0337; Co/sup 2 +/, 1.0283; Ni/sup 2 +/, 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 Mg/sup 2 +/ 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 /sup 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 /sup 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.
- Research Organization:
- Univ. of Wisconsin, Madison (USA)
- OSTI ID:
- 6999640
- Journal Information:
- Biochemistry; (United States), Vol. 27:8
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
HEMIACETAL DEHYDROGENASES
BIOCHEMICAL REACTION KINETICS
ISOTOPE EFFECTS
CADMIUM COMPOUNDS
CARBON 13
CATIONS
CHICKENS
COBALT COMPOUNDS
DECARBOXYLATION
DEUTERIUM
ENZYME ACTIVITY
LIVER
MAGNESIUM COMPOUNDS
MALIC ACID
MANGANESE COMPOUNDS
NADP
NICKEL COMPOUNDS
VISCOSITY
ZINC COMPOUNDS
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
BIRDS
BODY
CARBON ISOTOPES
CARBOXYLIC ACIDS
CHARGED PARTICLES
CHEMICAL REACTIONS
COENZYMES
DIGESTIVE SYSTEM
ENZYMES
EVEN-ODD NUCLEI
FOWL
GLANDS
HYDROGEN ISOTOPES
HYDROXY ACIDS
IONS
ISOTOPES
KINETICS
LIGHT NUCLEI
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
OXIDOREDUCTASES
REACTION KINETICS
STABLE ISOTOPES
TRANSITION ELEMENT COMPOUNDS
VERTEBRATES
550601* - Medicine- Unsealed Radionuclides in Diagnostics