Family 39 α-l -Iduronidases and β-d -Xylosidases React through Similar Glycosyl−Enzyme Intermediates: Identification of the Human Iduronidase Nucleophile
| Autor: | Shouming He, Stephen G. Withers, Catharine E. Nieman, Alexander W. Wong, Lorne A. Clarke, John J. Hopwood |
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| Rok vydání: | 2003 |
| Předmět: |
Iduronic Acid
Stereochemistry Molecular Sequence Data Glutamic Acid Bacillus Biochemistry Mass Spectrometry Iduronidase chemistry.chemical_compound Nucleophile Catalytic Domain Humans Glycoside hydrolase Glycosyl Amino Acid Sequence Conserved Sequence chemistry.chemical_classification Binding Sites Sequence Homology Amino Acid biology Active site Stereoisomerism Glutamic acid Xylosidases Peptide Fragments Kinetics Enzyme chemistry biology.protein Lactone |
| Zdroj: | Biochemistry. 42:8054-8065 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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