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

FEBS Letters

Evjen, Gry¹  Tollersrud, Ole K.¹  He, Shouming²  Numao, Shin²  Withers, Stephen G.²  Howard, Steven² 

[1] Department of Medical Biochemistry, University Hospital and University of Tromsø, Tromsø, Norway;Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C., Canada V6T 1Z1

关键词: Lysosomal α-mannosidase;    Nucleophile;    Mass spectrometry;    Enzyme mechanism;    Glycosidase;    5FgulF;    5-fluoro-β-L-gulopyranosyl fluoride;    DNP-Man;    2;    4-dinitrophenyl α-D-mannopyranoside;    ESI-MS;    electrospray ionisation mass spectrometry;    MS-MS;    tandem mass spectrometry;    CID;    collision-induced dissociation;   

DOI  :  10.1016/S0014-5793(00)02148-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown