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Identification of Glu-268 as the catalytic nucleophile of human lysosomal beta-galactosidase precursor by mass spectrometry

TitleIdentification of Glu-268 as the catalytic nucleophile of human lysosomal beta-galactosidase precursor by mass spectrometry
Publication TypeJournal Article
Year of Publication1997
AuthorsMCCARTER, JD, Burgoyne, DL, MIAO, SC, Zhang, SQ, Callahan, JW, Withers, SG
JournalJOURNAL OF BIOLOGICAL CHEMISTRY
Volume272
Pagination396-400
Date PublishedJAN 3
ISSN0021-9258
Abstract

Human lysosomal beta-galactosidase catalyzes the hydrolysis of beta-galactosides via a double displacement mechanism involving a covalent glycosyl enzyme intermediate. By use of the slow substrate 2,4-dinitrophenyl-2-deoxy-2-fluoro- beta-D-galactopyranoside, a glycosyl enzyme intermediate has been trapped on the enzyme. This has allowed the catalytic nucleophile to be identified as Glu-268 by peptic and tryptic digestion of the inactivated enzyme followed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry of the peptide mixture. This glutamic acid is fully conserved in a sequence-related family of enzymes (Family 35), consistent with its essential role.