Monitoring enzyme catalysis with mass spectrometry.
J Biol Chem
; 275(18): 13455-9, 2000 May 05.
Article
em En
| MEDLINE
| ID: mdl-10788458
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.
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Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
/
Enzimas
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2000
Tipo de documento:
Article