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A continuous, anaerobic spectrophotometric assay for chorismate synthase activity that utilizes photoreduced flavin
M K Ramjee1, J R Coggins, R N Thorneley
1AFRC Nitrogen Fixation Laboratory, University of Sussex, Brighton, United Kingdom.
Abstract:
A sensitive, continuous, spectrophotometric assay for chorismate synthase has been developed utilizing photoreduced flavin mononucleotide (FMNH2) as a cofactor under anaerobic conditions. The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at 275 nm with a precision of +/- 2 microM product. The assay conditions have been optimized with respect to FMNH2 (cofactor), EPSP (substrate) and enzyme concentrations, buffer type, and pH. The potential of the assay for detailed steady-state kinetic studies to elucidate the mechanism of action of this commercially important enzyme is also demonstrated.