Carbon-13 and deuterium isotope effects on oxalacetate decarboxylation by pyruvate carboxylase
Deuterium and /sup 13/C isotope effects for the enzymic decarboxylation of oxalacetate showed that both deuterium- and /sup 13/C-sensitive steps in the reaction are partially rate limiting. A normal alpha-secondary effect of 1.2 per deuterium was calculated for the reaction in which pyruvate-d3 was the substrate, suggesting that the enolate of pyruvate was an intermediate in the reaction. The large normal alpha-secondary deuterium isotope effect of 1.7 when oxalacetate-d2 was the substrate suggests that the motions of the secondary hydrogens are coupled to that of the primary hydrogen during the protonation of the enolate of pyruvate. The reduction in the magnitude of the /sup 13/C isotope effect for the oxamate-dependent decarboxylation of oxalacetate from 1.0238 to 1.0155 when the reaction was performed in D/sub 2/O (primary deuterum isotope effect = 2.1) clearly indicates that the transfer of the proton and carboxyl group between biotin and pyruvate does not occur via a single concerted reaction. Mechanisms in which biotin is activated to react with CO/sub 2/ (prior to transfer of the proton on N-1) by bond formation between the sulfur and the ureido carbon, or in which the sequence of events is decarboxylation of oxalacetate, proton transfer from biotin to enolpyruvate, and carboxylation of enolbiotin, predict that the /sup 13/C isotope effect in D/sub 2/O should be substantially lower than the observed value. A stepwise mechanism that does fit the data is one in which a proton is removed from biotin by a sulfhydryl group on the enzyme prior to carboxyl transfer, as long as the sulfhydryl group has an abnormally low pK.
- Research Organization:
- Univ. of Wisconsin, Madison
- OSTI ID:
- 6863304
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 25; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ACETATES
ANIMALS
BARYONS
BIOCHEMICAL REACTION KINETICS
BIRDS
BODY
CARBON 13
CARBON ISOTOPES
CARBON-CARBON LYASES
CARBOXY-LYASES
CARBOXYLASE
CARBOXYLIC ACID SALTS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHICKENS
DECARBOXYLATION
DEUTERIUM
DIGESTIVE SYSTEM
ELEMENTARY PARTICLES
ENZYMES
EVEN-ODD NUCLEI
FERMIONS
FOWL
GLANDS
HADRONS
HEAVY WATER
HYDROGEN COMPOUNDS
HYDROGEN ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPE EFFECTS
ISOTOPES
KETO ACIDS
KINETICS
LIGHT NUCLEI
LIVER
LYASES
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
OXYGEN COMPOUNDS
PROTONS
PYRUVIC ACID
REACTION KINETICS
STABLE ISOTOPES
TRACER TECHNIQUES
VERTEBRATES
WATER