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.
Numao
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Department of Chemistry, University of British Columbia, Vancouver, Canada.
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Evjen
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Howard
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Tollersrud
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Withers
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D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D020134:Catalytic Domain; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine; D043323:alpha-Mannosidase
D001224:Aspartic Acid; D008361:Mannosidases; D043323:alpha-Mannosidase