THE TRANSIENT KINETICS OF ESCHERICHIA-COLI CHORISMATE SYNTHASE - SUBSTRATE, CONSUMPTION, PRODUCT FORMATION, PHOSPHATE DISSOCIATION, AND CHARACTERIZATION OF A FLAVIN INTERMEDIATE

Chorismate synthase is the seventh enzyme of the shikimate pathway and catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The reaction involves the 1,4-elimination of phosphate and the C-(6proR) hydrogen of the substrate with unusual anti stereoc hemistry and requires a reduced flavin cofactor. This paper describes the kinetics of the formation and decay of a flavin intermediate, EPSP consumption, chorismate and phosphate formation, and phosphate dissoc iation during single and multiple turnover experiments, determined usi ng rapid reaction techniques. The kinetics of phosphate dissociation u sing the substrate analogues (6R)-[6-H-2]EPSP and (6S)-6-fluoro-EPSP h ave also been determined. The observations are consistent with a nonco ncerted chorismate synthase reaction. The flavin intermediate is not s imply associated with the conversion of substrate to product because i t forms before the substrate is consumed. The transient spectral chang es must be associated primarily with events such as protonation of the reduced flavin, a charge transfer complex between reduced flavin and an aromatic amino acid, or a conformational change in the protein. Thi s does not rule out the direct role of flavin in catalysis.