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Updated: Jul 18, 2026

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity
Published on: August 19, 2013
Mechanism-based inhibitors to probe transitional states of glycoside hydrolases
Hiroshi Hinou1, Masaki Kurogochi, Shin-Ichiro Nishimura
1Hokkaido University, Graduate School of Advanced Life Science, Sapporo, Japan.
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.
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