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 stereochemistry 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 dissociation during single and multiple turnover experiments, determined using rapid reaction techniques. The kinetics of phosphate dissociation using the substrate analogues (6R)-[6-H-2]EPSP and (6S)-6-fluoro-EPSP have also been determined. The observations are consistent with a nonconcerted chorismate synthase reaction. The flavin intermediate is not simply associated with the conversion of substrate to product because it forms before the substrate is consumed. The transient spectral changes 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. This does not rule out the direct role of flavin in catalysis.