Studies with substrate and cofactor analogues provide evidence for a radical mechanism in the chorismate synthase reaction

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-ph osphate (EPSP) to chorismate. The strict requirement for a reduced FMN cofa ctor and a trans-1,4-elimination are unusual. (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites i n the presence of dithionite. This conversion was associated with the oxida tion of FMN to give a stable flavin semiquinone. The IC,, of the fluorinate d substrate analogue was 0.5 and 250 muM with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not. The lack o f dissociation of the flavin semiquinone and chorismate from the enzyme app ears to be the basis of the essentially irreversible inhibition by this ana logue. A dithionite-dependent transient formation of flavin semiquinone dur ing turnover of (6S)-6-fluoro-EPSP has been observed. These reactions are b est rationalized by radical chemistry that is strongly supportive of a radi cal mechanism occurring during normal turnover. The lack of activity with 5 -deaza-FMN provides additional evidence for the role of flavin in catalysis by the E. coli enzyme.