Identification of Glu-268 as the catalytic nucleophile of human lysosomal beta-galactosidase precursor by mass spectrometry.
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.
McCarter
J D
JD
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Burgoyne
D L
DL
Miao
S
S
Zhang
S
S
Callahan
J W
JW
Withers
S G
SG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004792:Enzyme Precursors / Q000737:chemistry; D005733:Gangliosidoses / Q000201:enzymology; D005971:Glutamates / Q000737:chemistry; D006801:Humans; D008247:Lysosomes / Q000201:enzymology; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009598:Nitrophenylgalactosides / Q000494:pharmacology; D010449:Peptide Mapping; D016415:Sequence Alignment; D001616:beta-Galactosidase / Q000737:chemistry*
D004792:Enzyme Precursors; D005971:Glutamates; D009598:Nitrophenylgalactosides; D001616:beta-Galactosidase