title: Monitoring enzyme catalysis with mass spectrometry.
abstract: 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 alpha-glucosidase-catalyzed hydrolysis of p-nitrophenyl-alpha-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.
authors.affiliation: Scripps Research Institute, Beckman Center for Chemical Sciences, La Jolla, California 92037, USA.
authors.forename: B
authors.initials: B
authors.lastname: Bothner
authors.forename: R
authors.initials: R
authors.lastname: Chavez
authors.forename: J
authors.initials: J
authors.lastname: Wei
authors.forename: C
authors.initials: C
authors.lastname: Strupp
authors.forename: Q
authors.initials: Q
authors.lastname: Phung
authors.forename: A
authors.initials: A
authors.lastname: Schneemann
authors.forename: G
authors.initials: G
authors.lastname: Siuzdak
mesh_terms: D000818:Animals; D002384:Catalysis; D004798:Enzymes / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D004798:Enzymes