|Title||Identification of Glu-268 as the catalytic nucleophile of human lysosomal beta-galactosidase precursor by mass spectrometry|
|Publication Type||Journal Article|
|Year of Publication||1997|
|Authors||MCCARTER, JD, Burgoyne, DL, MIAO, SC, Zhang, SQ, Callahan, JW, Withers, SG|
|Journal||JOURNAL OF BIOLOGICAL CHEMISTRY|
|Date Published||JAN 3|
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.