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Type: Journal article
Title: Family 39 a-L-iduronidases and b-D-xylosidases react through similar glycosyl-enzyme intermediates: Identification of the human iduronidase nucleophile
Author: Nieman, C.
Wong, A.
He, S.
Clarke, L.
Hopwood, J.
Withers, S.
Citation: Biochemistry, 2003; 42(26):8054-8065
Publisher: Amer Chemical Soc
Issue Date: 2003
ISSN: 0006-2960
Abstract: The inclusion of both beta-D-xylosidases and alpha-L-iduronidases within the same sequence-related family (family 39), despite the considerable difference in substrate structures and poor sequence conservation around the putative nucleophile, raises concerns about whether a common mechanism is followed by the two enzymes. A novel anchimeric assistance mechanism for iduronidases involving a lactone intermediate is one possibility. NMR analysis of the methanolysis reaction catalyzed by human alpha-L-iduronidase reveals that, as with the beta-D-xylosidases, alpha-L-iduronidase is a retaining glycosidase. Using two different mechanism-based inactivators, 5-fluoro-alpha-L-iduronyl fluoride and 2-deoxy-2-fluoro-alpha-L-iduronyl fluoride, the active site nucleophile in the human alpha-L-iduronidase was identified as Glu299 within the (295)IYNDEAD(301) sequence. The equivalent, though loosely predicted, glutamic acid was identified as the nucleophile in the family 39 beta-D-xylosidase from Bacillus sp. [Vocadlo, D., et al. (1998) Biochem. J. 335, 449-455]; thus, a common mechanism involving a covalent glycosyl-enzyme intermediate that adopts the rather uncommon (2,5)B conformation is predicted.
Keywords: Humans
Iduronic Acid
Glutamic Acid
Peptide Fragments
Binding Sites
Amino Acid Sequence
Catalytic Domain
Conserved Sequence
Sequence Homology, Amino Acid
Molecular Sequence Data
Mass Spectrometry
DOI: 10.1021/bi034293v
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