Identification of the catalytic nucleophile in Family 42 beta-galactosidases by intermediate trapping and peptide mapping: YesZ from Bacillus subtilis.
FEBS Lett
; 581(13): 2441-6, 2007 May 29.
Article
en En
| MEDLINE
| ID: mdl-17485082
ABSTRACT
The mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-galactopyranoside (DNP2FGal) was used to inactivate the Family 42 beta-galactosidase (YesZ) from Bacillus subtilis via the trapping of a glycosyl-enzyme intermediate, thereby tagging the catalytic nucleophile in the active site. Proteolytic digestion of the inactivated enzyme and of a control sample of unlabeled enzyme, followed by comparative high-performance liquid chromatography and mass spectrometric analysis identified a labelled peptide of the sequence ETSPSYAASL. These data, combined with sequence alignments of this region with representative members of Family 42, unequivocally identify the catalytic nucleophile in this enzyme as Glu-295, thereby providing the first direct experimental proof of the identity of this residue within Family 42.
Buscar en Google
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Bacillus subtilis
/
Proteínas Bacterianas
/
Beta-Galactosidasa
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
FEBS Lett
Año:
2007
Tipo del documento:
Article
País de afiliación:
Canadá