Identification of 2-enolbutyrate as the product of the reaction of maize leaf phosphoenolpyruvate carboxylase with (Z)- and (E)-2-phosphoenolbutyrate: evidence from NMR and kinetic measurements
(Z)- and (E)-2-phosphoenolbutyrates were dephosphorylated at similar rates by phosphoenolpyruvate carboxylase purified from maize leaves, as determined from proton nuclear magnetic resonance measurements. The product of the reaction in D/sub 2/O was a mixture of 60-70% 2-oxo(3-H,D)butyrate, 25-30% 2-oxo(3-D/sub 2/) butyrate, and 5-10% 2-oxo(3-H/sub 2/) butyrate. The amounts of (R)- and (S)-2-oxo(3-H,D) butyrate in this mixture were determined by exchange at C-3 in D/sup 2/O catalyzed by pyruvate kinase as described previously. Forty-five minutes after the addition of pyruvate kinase, the proportions of 2-oxo(3-H,D) butyrate and 2-oxo(3-D/sub 2/)-butyrate were 36-39% and 61-64%, respectively, indicating that the original mixture contained equal amounts of R and S enantiomers. In addition, a compound with properties similar to those of enolpyruvate was detected in solution during the action of phosphoenolpyruvate carboxylase on 2-phosphoenolbutyrate. This compound, most likely 2-enolbutyrate, presented maximum light absorption at 220-230 nm and was ketonized in a solution containing 80% D/sub 2/O and 20% H/sub 2/O (pH 7) with a rate constant of 1.33 min/sup -1/. From these results, it is concluded that the actual product released from the active site of phosphoenolpyruvate carboxylase during the reaction with 2-phosphoenolbutyrate is the enolic form of 2-oxobutyrate and that protonation of this form takes place at random in solution.
- Research Organization:
- CEFOBL, Rosario, Argentina
- OSTI ID:
- 5355175
- Journal Information:
- Biochemistry; (United States), Vol. 27:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
BUTYRIC ACID
BIOCHEMICAL REACTION KINETICS
NUCLEAR MAGNETIC RESONANCE
CARBOXY-LYASES
CARBOXYLATION
DEUTERIUM COMPOUNDS
ENANTIOMORPHS
HEAVY WATER
LEAVES
MAIZE
NMR SPECTRA
PHOSPHOTRANSFERASES
PROTONS
PYRUVIC ACID
BARYONS
CARBON-CARBON LYASES
CARBOXYLIC ACIDS
CEREALS
CHEMICAL REACTIONS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
GRASS
HADRONS
HYDROGEN COMPOUNDS
KETO ACIDS
KINETICS
LYASES
MAGNETIC RESONANCE
MONOCARBOXYLIC ACIDS
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PLANTS
REACTION KINETICS
RESONANCE
SPECTRA
TRANSFERASES
WATER
550601* - Medicine- Unsealed Radionuclides in Diagnostics