Identification of Glu-268 as the Catalytic Nucleophile of Human Lysosomal β-Galactosidase Precursor by Mass Spectrometry

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.