Mechanistic investigation of the endo-alpha-N-acetylgalactosaminidase from Streptococcus pneumoniae R6.
The large (1767-amino acid) endo-alpha-N-acetylgalactosaminidase from Streptococcus pneumoniae (SpGH101) specifically removes an O-linked disaccharide Gal-beta-1,3-GalNAc-alpha 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 alpha-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. pK(a) values of 5.3 and 7.2 were assigned to D764 and E796 on the basis of the pK(a) values derived from the bell-shaped dependence of k(cat)/K(m) 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-N-biose, consistent with a role for these domains in localizing the enzyme near its substrates.
Glycobiology Program, Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario, Canada K1A 0R6.
Lisa M
LM
Willis
Ran
R
Zhang
Anne
A
Reid
Stephen G
SG
Withers
Warren W
WW
Wakarchuk
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D048809:alpha-N-Acetylgalactosaminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; D048809:alpha-N-Acetylgalactosaminidase