Mechanistic investigation of the endo-α-Ν-acetylgalactosaminidase from Streptococcus pneumoniae R6

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DOIResolve DOI: http://doi.org/10.1021/bi9013825
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TypeArticle
Journal titleBiochemistry
Volume48
Issue43
Pages1033410341; # of pages: 8
AbstractThe large (1767-amino acid) endo-α-Ν-acetylgalactosaminidase from Streptococcus pneumoniae (SpGH101) specifically removes an O-linked disaccharide Gal-β-1,3-GalNAc-α from glycoproteins. While the enzyme from natural sources has been used as a reagent for many years, very few mechanistic studies have been performed. Using the recently determined three-dimensional structure of the recombinant protein as a background, we report here a mechanistic investigation of the SpGH101 retaining R-glycoside hydrolase using a combination of synthetic and natural substrates. On the basis of a model of the substrate complex of SpGH101, we propose D764 and E796 as the nucleophile and general acid-base residues, respectively. These roles were confirmed by kinetic and mechanistic analysis of mutants at those positions using synthetic substrates and anion rescue experiments. pKa values of 5.3 and 7.2 were assigned to D764 and E796 on the basis of the pKa values derived from the bell-shaped dependence of kcat/Km upon pH. The enzyme contains several putative carbohydrate binding modules whose glycan binding specificities were probed using the printed glycan array of the Consortium for Functional Glycomics using the inactive D764A and D764F mutants that had been labeled with Alexafluor 488. These studies revealed binding to galacto-Ν-biose, consistent with a role for these domains in localizing the enzyme near its substrates.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); NRC Institute for Biological Sciences
Peer reviewedYes
NPARC number15295293
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Record identifier623e8327-5e29-40a0-8276-bf9721ab3334
Record created2010-05-17
Record modified2016-05-09
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