Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/7022
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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
1520-4995
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
Bacillus
Iduronic Acid
Iduronidase
Xylosidases
Glutamic Acid
Peptide Fragments
Binding Sites
Amino Acid Sequence
Catalytic Domain
Conserved Sequence
Sequence Homology, Amino Acid
Kinetics
Stereoisomerism
Molecular Sequence Data
Mass Spectrometry
DOI: 10.1021/bi034293v
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