Ramjee M K, Coggins J R, Thorneley R N
AFRC Nitrogen Fixation Laboratory, University of Sussex, Brighton, United Kingdom.
Anal Biochem. 1994 Jul;220(1):137-41. doi: 10.1006/abio.1994.1309.
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.
已开发出一种灵敏、连续的分光光度法测定分支酸合酶，该方法利用光还原黄素单核苷酸（FMNH₂）作为辅因子，在厌氧条件下进行。该测定法直接监测在275nm处由5-烯醇丙酮酸莽草酸-3-磷酸（EPSP）形成分支酸的过程，产物的测定精度为±2μM。已针对FMNH₂（辅因子）、EPSP（底物）和酶浓度、缓冲液类型及pH对测定条件进行了优化。还展示了该测定法用于详细稳态动力学研究以阐明这种具有商业重要性的酶的作用机制的潜力。
