W. Bondinell, J. Vnek, P. Knowles
Oct 25, 1971
Citations
1
Influential Citations
67
Citations
Journal
The Journal of biological chemistry
Abstract
Abstract The enzymic synthesis of 5-enolpyruvylshikimate 3-phosphate (ES-3-P) from enolpyruvate phosphate (labeled with 18O in the C-O-P oxygen) and shikimate 3-phosphate occurred with C-O cleavage of the pyruvate ester. ES-3-P formed in a D2O reaction medium showed incorporation of approximately 1.3 atoms of deuterium, and consisted of 39% d2 molecules, 53% d1, and 8% d0. Enolpyruvate phosphate isolated from the same reaction mixture had 0.72 atom of deuterium with a similar d2:d1 ratio. In both compounds, the nuclear magnetic resonances of the vinyl methylene protons were diminished equally in intensity. ES-3-P formed in a tritiated medium contained 0.42 atom of 3H in the vinyl methylene hydrogens. A reversible addition-elimination mechanism is proposed in which protonation of carbon 3 of enolpyruvate phosphate is associated with a nucleophilic attack on carbon 2 by the 5-hydroxyl group of shikimate 3-phosphate. A methyl group of unrestricted rotation is formed in the resulting postulated intermediate. Elimination of orthophosphate then yields ES-3-P, and elimination of shikimate 3-phosphate yields enolpyruvate phosphate. A procedure was developed for preparing enolpyruvate phosphate with 18O in the C-O-P oxygen. The isolation of shikimate 3-phosphate was improved. ES-3-P synthetase from Salmonella was purified 80-fold by a simple procedure.