Often glycosidase assays are based on small-molecule compounds where a glycan of interest is linked to a chromophore allowing for easy detection of cleavage of the glycoside bond. However, such compounds only resemble part of the more complex substrate molecule for enzymes acting on glycoconjugates of glycopeptides or glycoproteins. Nonetheless, the advantage is obvious as enzyme activity is readily recorded and kinetic parameters easily obtained. This is not often the case with glycopeptides or glycoproteins as these may reveal increased complexity in terms of heterogeneity in protein-glycan stoichiometry and restricted enzyme accessibility. However, a kinetic analysis of glycan release from glycopeptides could provide information complementary to that of small-molecule substrates, especially if providing kinetic parameters that are immediately comparable. We have characterized the steady state kinetics of wild type and mutant variants of Bifidobacterium longum endo-α-N-acetylgalactosaminidase, by recording the enzymatic release of Galβ(1-3)GalNAc from bovine glycomacropeptide pre-treated with sialidase to remove sialic acid units. Differences between previously reported kinetic constants obtained with synthetic substrates and those obtained in the present work demonstrate an influence of the peptide moiety on the kinetic properties of endo-α-N-acetylgalactosaminidase. The devised assay and data handling method determines the accessible substrate concentration as well as the steady state kinetic parameters, KM and kcat, for glycoconjugates of glycopeptides described by the same units as obtained from using small-molecule substrates and thus allows for a direct comparison.
Keywords: 2-N-acetyl galactose; Asialo-CGMP; CGMP; CGMP treated to reaction completion with NanI; DMAB; EngBF, EngSP and EngCP; GH101; Gal; GalNAc; Glycanase; IPTG; Isopropyl β-d-1-thiogalactopyranoside; Morgan-Elson; NanI; NanI sialidase from Clostridium perfringens; O-deglycosylation; T antigen; endo-α-N-acetylgalactosaminidase from Bifidobacterium longum, Streptococcus pneumoniae and Clostridium perfringens, respectively; galactosyl; para-dimethylaminobenzaldehyde; κ-casein derived glycomacropeptide.
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