Monitoring enzyme catalysis with mass spectrometry.
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.
Bothner
B
B
Scripps Research Institute, Beckman Center for Chemical Sciences, La Jolla, California 92037, USA.
Chavez
R
R
Wei
J
J
Strupp
C
C
Phung
Q
Q
Schneemann
A
A
Siuzdak
G
G
D000818:Animals; D002384:Catalysis; D004798:Enzymes / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*
D004798:Enzymes