Abstract
Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The strict requirement for a reduced FMN cofactor and a trans-1,4-elimination are unusual. (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites in the presence of dithionite. This conversion was associated with the oxidation of FMN to give a stable flavin semiquinone. The IC(50) of the fluorinated substrate analogue was 0.5 and 250 microm with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not. The lack of dissociation of the flavin semiquinone and chorismate from the enzyme appears to be the basis of the essentially irreversible inhibition by this analogue. A dithionite-dependent transient formation of flavin semiquinone during turnover of (6S)-6-fluoro-EPSP has been observed. These reactions are best rationalized by radical chemistry that is strongly supportive of a radical 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.
Highlights
Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP)1 to chorismate (Scheme 1) [1]
This paper describes new studies with the E. coli enzyme that are strongly supportive of the proposed radical mechanism (Scheme 1A)
1b) led to the formation of a neutral flavin semiquinone radical (max 584 nm) that reached a maximum after about 4 min
Summary
Materials—All chemicals and biochemicals were of the highest grade available and, unless stated otherwise, were purchased from Sigma. The potassium salt of EPSP was prepared as described previously [17]. Enzyme Purification and UV Assay—Recombinant E. coli chorismate synthase was purified as reported previously [17], and enzyme concentration was determined using ⑀280 ϭ 21,440 MϪ1 cmϪ1 per subunit [20]. The enzyme was assayed directly by UV spectrophotometry as described previously [21], and substrate concentrations were determined using this method. One unit of enzyme is defined as the amount of enzyme required to convert 1 mol of substrate to product in 1 min.
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