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

Abstract

Bovine kidney lysosomal α-mannosidase is a family 38 α-mannosidase involved in the degradation of glycoproteins. The mechanism-based reagent, 5-fluoro-β- 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 α-mannosidases of the same family, this result strongly suggests that the catalytic nucleophile in this enzyme is Asp197.