A continuous, anaerobic spectrophotometric assay for chorismate synthase activity that utilizes photoreduced flavin mononucleotide.
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.
AFRC Nitrogen Fixation Laboratory, University of Sussex, Brighton, United Kingdom.
M K
MK
Ramjee
J R
JR
Coggins
R N
RN
Thorneley
D000693:Anaerobiosis; D004926:Escherichia coli / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000737:chemistry*; D007700:Kinetics; D008190:Lyases / Q000032:analysis*; D010084:Oxidation-Reduction; D019761:Phosphorus-Oxygen Lyases; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet
D005486:Flavin Mononucleotide; D008190:Lyases; C019895:chorismate synthase; D019761:Phosphorus-Oxygen Lyases