Kinetics of an enzyme-catalyzed reaction measured by electrospray ionization mass spectrometry using a simple rapid mixing attachment.
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 N2-(carbobenzyloxy)-L-lysine-p-nitrophenyl 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.
Attwood
Paul V
PV
Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, The University of Western Australia, 35, Stirling Highway, Crawley, WA 6100, Australia. pattwood@cyllene.uwa.edu.au
Geeves
Michael A
MA
D002384:Catalysis; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation* / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism*
C024992:N(alpha)-carbobenzoxylysine-4-nitroanilide; D014357:Trypsin; D008239:Lysine