The Transient Kinetics of Escherichia coli Chorismate Synthase: Substrate Consumption, Product Formation, Phosphate Dissociation, and Characterization of a Flavin Intermediate

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• 作者：Stephen Bornemann ; David J. Lowe ; and Roger N. F. Thorneley
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：July 30, 1996
• 年：1996
• 卷：35
• 期：30
• 页码：9907 - 9916
• 全文大小：497K
• 年卷期：v.35,no.30(July 30, 1996)
• ISSN：1520-4995

文摘

Chorismate synthase is the seventh enzyme of the shikimate pathwayand catalyzes theconversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate.The reaction involves the 1,4-elimination of phosphate and the C-(6proR) hydrogen of thesubstrate with unusual anti stereochemistryand requires a reduced flavin cofactor. This paper describes thekinetics of the formation and decay ofa flavin intermediate, EPSP consumption, chorismate and phosphateformation, and phosphate dissociationduring single and multiple turnover experiments, determined using rapidreaction techniques. The kineticsof phosphate dissociation using the substrate analogues(6R)-[6-²H]EPSP and(6S)-6-fluoro-EPSP havealso been determined. The observations are consistent with anonconcerted chorismate synthase reaction.The flavin intermediate is not simply associated with theconversion of substrate to product because itforms before the substrate is consumed. The transient spectralchanges must be associated primarily withevents such as protonation of the reduced flavin, a charge transfercomplex between reduced flavin andan aromatic amino acid, or a conformational change in the protein.This does not rule out the direct roleof flavin in catalysis.