A Continuous, Anaerobic Spectrophotometric Assay for Chorismate Synthase Activity That Utilizes Photoreduced Flavin Mononucleotide

✍ Scribed by M.K. Ramjee; J.R. Coggins; R.N.F. Thorneley

• Publisher: Elsevier Science
• Year: 1994
• Tongue: English
• Weight: 364 KB
• Volume: 220
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1994.1309

✦ Synopsis

A sensitive, continuous, spectrophotometric assay for chorismate synthase has been developed utilizing photoreduced flavin mononucleotide (\left(\mathbf{F M N H}{2}\right.) ) as a cofactor under anaerobic conditions. The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at (275 \mathrm{~nm}) with a precision of (\pm 2 \mu \mathrm{M}) product. The assay conditions have been optimized with respect to (\mathrm{FMNH}{2}) (cofactor), EPSP (substrate) and enzyme concentrations, buffer type, and (\mathrm{pH}). The potential of the assay for detailed steadystate kinetic studies to elucidate the mechanism of action of this commercially important enzyme is also demonstrated. iç 1994 Academic Press, Inc.