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Identification of the catalytic nucleophile in Family 42 β‐galactosidases by intermediate trapping and peptide mapping: YesZ from Bacillus subtilis
Author(s) -
Shaikh Fathima Aidha,
Müllegger Johannes,
He Shouming,
Withers Stephen G.
Publication year - 2007
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2007.04.053
Subject(s) - bacillus subtilis , chemistry , nucleophile , enzyme , residue (chemistry) , stereochemistry , peptide , galactosidases , proteolytic enzymes , peptide sequence , biochemistry , catalysis , beta galactosidase , biology , escherichia coli , genetics , bacteria , gene
The mechanism‐based inhibitor 2,4‐dinitrophenyl 2‐deoxy‐2‐fluoro‐β‐ d ‐galactopyranoside (DNP2FGal) was used to inactivate the Family 42 β‐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.