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J Biol Chem, Vol. 275, Issue 18, 13455-13459, May 5, 2000

Monitoring Enzyme Catalysis with Mass Spectrometry^*

Brian Bothner, Rodrigo Chavez, Jing Wei, Christian Strupp, Qui Phung, Anette Schneemann, and Gary Siuzdak^§

From The Scripps Research Institute, Beckman Center for Chemical Sciences, La Jolla, California 92037 and  Mass Consortium Corporation, San Diego, California 92121

Mass spectrometry is a rapid, sensitive, and accurate quantitative approach for the direct monitoring of enzyme-catalyzed reactions that does not require a chromophore or radiolabeling and thus provides a viable alternative to existing analytical techniques. In this study the proteolysis of intact viral capsid proteins, the -glucosidase-catalyzed hydrolysis of p-nitrophenyl--glucopyranoside and the lipoprotein lipase-catalyzed ester hydrolysis of resorufin were examined. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry were used to examine the proteolysis of viral protein capsids, providing information about capsid dynamics and the stabilizing force of viral protein/RNA interactions. In addition, k_cat and K_m values of enzyme-catalyzed hydrolysis were obtained (without the use of a chromophore). These results also demonstrate the effect an unnatural substrate can have on enzyme activity. Overall, mass spectrometry provides for efficient and quantitative analysis of enzyme-catalyzed reactions, as well as the direct observation of reaction dynamics.

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