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Mechanism of the EPSP synthase catalyzed reaction: evidence for the lack of a covalent carboxyvinyl intermediate in catalysis

Journal Article · · Biochem. Biophys. Res. Commun.; (United States)
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In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP. An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D/sub 2/O. Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+. Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP. No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex. Incorporation of (/sup 14/C) from (/sup 14/C)-S3P and (/sup 14/C)-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system. This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange.

Research Organization:
Biological Sciences, Monsanto, Chesterfield, MO (USA)
OSTI ID:
6924755
Journal Information:
Biochem. Biophys. Res. Commun.; (United States), Journal Name: Biochem. Biophys. Res. Commun.; (United States) Vol. 153:2; ISSN BBRCA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BARYONS
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CATALYSIS
DEUTERIUM
ELEMENTARY PARTICLES
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
FERMIONS
HADRONS
HYDROGEN ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGASES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
OXYGEN COMPOUNDS
PHOSPHATES
PHOSPHORUS COMPOUNDS
PROTONS
REACTION KINETICS
RESONANCE
STABLE ISOTOPES
TRACER TECHNIQUES
TRANSFERASES