title: The identification of chemical intermediates in enzyme catalysis by the rapid quench-flow technique.
abstract: Traditionally, enzyme transient kinetics have been studied by the stopped-flow and rapid quench-flow (QF) methods. Whereas stopped-flow is the more convenient, it suffers from two weaknesses: optically silent systems cannot be studied, and when there is a signal it cannot always be assigned to a particular step in the reaction pathway. QF is a chemical sampling method; reaction mixtures are aged for a few milliseconds or longer, 'stopped' by a quenching agent and the product or the intermediate is measured by a specific analytical method. Here we show that by exploiting the array of current analytical methods and different quenching agents, the QF method is a key technique for identifying, and for characterising kinetically, intermediates in enzyme reaction pathways and for determining the order by which bonds are formed or cleaved by enzymes acting on polymer substrates such as DNA.
authors.affiliation: UMR 5121, CNRS-University Montpellier I, Institut de Biologie, 4 bd Henri IV, 34000 Montpellier, France.
authors.forename: T E
authors.initials: TE
authors.lastname: Barman
authors.forename: S R W
authors.initials: SR
authors.lastname: Bellamy
authors.forename: H
authors.initials: H
authors.lastname: Gutfreund
authors.forename: S E
authors.initials: SE
authors.lastname: Halford
authors.forename: C
authors.initials: C
authors.lastname: Lionne
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009218:Myosins / Q000737:chemistry / Q000378:metabolism; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism
chemical_list: D004798:Enzymes; D000255:Adenosine Triphosphate; D004247:DNA; D010770:Phosphotransferases; D009218:Myosins