Skip to main content
Cellular and Molecular Life Sciences: CMLS logoLink to Cellular and Molecular Life Sciences: CMLS
. 2006 Sep 4;63(22):2571–2583. doi: 10.1007/s00018-006-6243-z

The identification of chemical intermediates in enzyme catalysis by the rapid quench-flow technique

T E Barman 1, S R W Bellamy 2, H Gutfreund 2, S E Halford 2, C Lionne 1,
PMCID: PMC11136296  PMID: 16952048

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.

Keywords. Enzyme mechanism, protein dynamics, rapid-reaction technique, kinetics, ATPase, DNA-protein interaction

Footnotes

Received 24 May 2006; received after revision: 3 July 2006; accepted 19 July 2006


Articles from Cellular and Molecular Life Sciences: CMLS are provided here courtesy of Springer

RESOURCES