Kinetics of an enzyme-catalyzed reaction measured by electrospray ionization mass spectrometry using a simple rapid mixing attachment

Paul Attwood, M.A. Geeves

Research output: Contribution to journalArticlepeer-review

22 Citations (Scopus)

Abstract

Mass spectrometry offers a potential means of measuring virtually all enzyme-catalyzed reactions by simultaneously measuring the concentrations of substrates, products, and intermediates where there are differences in mass between them. To perform these measurements the reaction mixture must be aged for different times and then ionized. Electrospray ionization mass spectrometry provides the most direct means of measuring these reactions. Here we describe a simple reaction mixing and ageing attachment for an electrospray ionization mass spectrometer, built from commercially available components. We have employed this device to measure the kinetics of a model reaction, namely the hydrolysis of N-2-(carbobenzyloxy)-L-lysine-p-nitrophenyI ester-catalyzed by trypsin. In this way we were able to measure the kinetics of substrate depletion, product formation, and changes in both free enzyme and acyl-enzyme intermediate concentration in the approach to steady state. With this device we were able to measure reaction times down to about 640 ms. (C) 2004 Elsevier Inc. All rights reserved.
Original languageEnglish
Pages (from-to)382-389
JournalAnalytical Biochemistry
Volume334
Issue number2
DOIs
Publication statusPublished - 2004

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