Syntheses of model compounds related to an antigenic epitope in pectic polysaccharides from Bupleurum falcatum L |
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Authors: | Hada N Ogino T Yamada H Takeda T |
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Institution: | Department of Chemistry, Protein Engineering Network of Centres of Excellence of Canada, University of British Columbia, Vancouver, BC, Canada V6T 1Z1. |
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Abstract: | The beta-galactosidases from Xanthomonas manihotis (beta-Gal Xmn) and Bacillus circulans (beta-Gal-3 Bcir) are retaining glycosidases that hydrolyze glycosidic bonds through a double displacement mechanism involving a covalent glycosyl-enzyme intermediate. The mechanism-based inactivator 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside was shown to inactivate beta-Gal Xmn and beta-Gal-3 Bcir through the accumulation of 2-deoxy-2-fluorogalactosyl enzyme intermediates with half lives of 40 and 625 h, respectively. Peptic digestion of these labeled enzymes and analysis by LC-MS identified Glu(260) and Glu(233) as the catalytic nucleophiles involved in the formation of the glycosyl-enzyme intermediate during catalysis by beta-Gal Xmn and beta-Gal-3 Bcir, respectively. These findings confirm the previous prediction of the position of these residues based on primary sequence similarities to other members of the glycoside hydrolase family 35. |
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