title: Mechanism-based inhibitors to probe transitional states of glycoside hydrolases.
abstract: Recent structural and kinetic studies indicate that glycosidases (glycoside hydrolases) change the peripheral structure of their catalytic sites dynamically to trim glycan structures. Inhibitors that label specific amino acid residues in the active site of these enzymes based on its mechanism of action are powerful tools to probe such a hidden transitional state. This chapter describes methods of mechanism-based irreversible inhibitors having fluorescence tags, including synthesis, inhibitory assay, rapid separation of the peptides containing labeled residues using antibody column, and proteomic analysis of key amino acid residues using matrix-assisted laser desorption/ionization-time-of-flight (TOF)/TOF mass spectrometry.
authors.affiliation: Hokkaido University, Graduate School of Advanced Life Science, Sapporo, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hinou
authors.forename: Masaki
authors.initials: M
authors.lastname: Kurogochi
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D005456:Fluorescent Dyes / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D008958:Models, Molecular; D015394:Molecular Structure; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014734:Vibrio cholerae / Q000201:enzymology
chemical_list: D005456:Fluorescent Dyes; D011134:Polysaccharides; D006026:Glycoside Hydrolases; D009439:Neuraminidase