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Title: Observation by sup 13 C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site

Direct observation of the tetrahedral intermediate in the EPSP synthase reaction pathway was provided by {sup 13}C NMR by examining the species bound to the enzyme active site under internal equilibrium conditions and using (2-{sup 13}C)PEP as a spectroscopic probe. The tetrahedral center of the intermediate bound to the enzyme gave a unique signal appearing at 104 ppm. Separate signals were observed for free EPSP and EPSP bound to the enzyme in a ternary complex with phosphate. These peak assignments account for the quantitation of the species bound to the enzyme and liberated upon quenching with either triethylamine or base. A comparison of quenching with acid, base, or triethylamine was conducted. After long times of incubation during the NMR measurement, a signal at 107 ppm appeared. The compound giving rise to this resonance was isolated and identified as an EPSP ketal. The rate of formation of the EPSP ketal was very slow establishing that it is a side product of the normal enzymatic reaction. To look for additional signals that might arise from a covalent adduct which has been postulated to arise from reaction of enzyme with PEP, and NMR experiment was performed with an analogue of S3P lacking themore » 4- and 5-hydroxyl groups. All of these results reaffirm identification of the tetrahedral species as the only observable intermediate in the EPSP synthase reaction.« less
Authors:
; ; ;  [1] ; ;  [2]
  1. (Monsanto Agricultural Company, St. Louis, MO (USA))
  2. (Pennsylvania State Univ., University Park (USA))
Publication Date:
OSTI Identifier:
7100660
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (USA); Journal Volume: 29:6
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; LIGASES; NUCLEAR MAGNETIC RESONANCE; SHIKIMIC ACID; BIOLOGICAL PATHWAYS; CARBON 13; LIQUID COLUMN CHROMATOGRAPHY; NMR SPECTRA; PHOSPHOENOLPYRUVATE; CARBON ISOTOPES; CARBOXYLIC ACIDS; CHROMATOGRAPHY; ENZYMES; EVEN-ODD NUCLEI; HYDROXY ACIDS; ISOTOPES; LIGHT NUCLEI; MAGNETIC RESONANCE; NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; RESONANCE; SEPARATION PROCESSES; SPECTRA; STABLE ISOTOPES 550601* -- Medicine-- Unsealed Radionuclides in Diagnostics