Isotope effect studies of the chemical mechanism of pig heart NADP isocitrate dehydrogenase
The catalytic mechanism of porcine heart NADP isocitrate dehydrogenase has been investigated by use of the variation of deuterium and /sup 13/C kinetic isotope effects with pH. The observed /sup 13/C isotope effect on VK for isocitrate increases from 1.0038 at neutral pH to a limiting value of 1.040 at low pH. The limiting /sup 13/C isotope effect with deuteriated isocitrate at low pH is 1.016. This decrease in /sup 13/(VK/sub Ic/) upon deuteriation indicates a stepwise mechanism for the oxidation and decarboxylation of isocitrate. This predicts a deuterium isotope effect on VK of 2.9, but /sup D/(VK) at low pH only increases to a maximum of 1.08. The pK seen in the /sup 13/(VK/sub Ic/) pH profile for isocitrate if 4.5. This pK is displaced 1.2 pH units from the true pK of the acidbase functionality of 5.7 seen in the pK/sub i/ profile for oxalylglycine. From this displacement, catalysis is estimated to be 16 times faster than substrate dissociation. By use of the pH-dependent partitioning ratio of the reaction intermediate oxalosuccinate between decarboxylation to 2-ketoglutarate and reduction to isocitrate, the forward commitment to catalysis for decarboxylation was determined to be 7.3 at pH 5.4 and 3.2 at pH 5.0. This gives in intrinsic /sup 13/C isotope effect for decarboxylation of 1.050. The product of oxidative decarboxylation of 3-hydroxyisocitrate by NADP isocitrate dehydrogenase is 2-hydroxy-3-ketoglutarate. This results from enzymatic protonation of the cis-enediol intermediate at C/sub 2/ rather than C/sub 3/ (as seen with isocitrate and 3-fluoroisocitrate). 2-Hydroxy-3-ketoglutarate further decarboxylates in solution to 2-hydroxy-3-ketobutyrate, which further decarboxylates to acetol. This makes 3-hydroxyisocitrate unsuitable for /sup 13/C isotope effect studies.
- Research Organization:
- Univ. of Wisconsin, Madison (USA)
- OSTI ID:
- 6999636
- 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
CARBON 13
CITRATES
DECARBOXYLATION
DEUTERIUM
HEART
NADP
NUCLEAR MAGNETIC RESONANCE
PH VALUE
SWINE
VISCOSITY
ANIMALS
BODY
CARBON ISOTOPES
CARBOXYLIC ACID SALTS
CARDIOVASCULAR SYSTEM
CHEMICAL REACTIONS
COENZYMES
DOMESTIC ANIMALS
ENZYMES
EVEN-ODD NUCLEI
HYDROGEN ISOTOPES
ISOTOPES
KINETICS
LIGHT NUCLEI
MAGNETIC RESONANCE
MAMMALS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
OXIDOREDUCTASES
REACTION KINETICS
RESONANCE
STABLE ISOTOPES
VERTEBRATES
550601* - Medicine- Unsealed Radionuclides in Diagnostics