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-ga lactosides via a double displacement mechanism involving a covalent gl ycosyl enzyme intermediate. By use of the slow substrate 2,4-dinitroph enyl-2-deoxy-2-fluoro- beta-D-galactopyranoside, a glycosyl enzyme int ermediate has been trapped on the enzyme. This has allowed the catalyt ic nucleophile to be identified as Glu-268 by peptic and tryptic diges tion of the inactivated enzyme followed by high performance liquid chr omatography-electrospray ionization tandem mass spectrometry of the pe ptide mixture. This glutamic acid is fully conserved in a sequence-rel ated family of enzymes (Family 35), consistent with its essential role .