Identification of Asp197 as the catalytic nucleophile in the family 38 alpha-mannosidase from bovine kidney lysosomes

Bovine kidney lysosomal alpha -mannosidase is a family 38 alpha -mannosidase involved in the degradation of glycoproteins. The mechanism-based reagent, 5-fluoro-beta -L-gulosyl fluoride, was used to trap a glycosyl-enzyme intermediate, thereby labelling the catalytic nucleophile of this enzyme. After proteolytic digestion and high performance liquid chromatography/tandem mass spectrometry (MS) analysis, a labelled peptide was localised, and the sequence: HIDPFGHSRE determined by fragmentation tandem MS analysis, Taking into consideration sequence alignments of this region with those of other alpha -mannosidases of the same family, this result strongly suggests that the catalytic nucleophile in this enzyme is Asp197. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B,V, All rights reserved.