Journal of Biomolecular NMR

, Volume 67, Issue 4, pp 309–319 | Cite as

NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

  • Shoko Shinya
  • Mariana G. Ghinet
  • Ryszard Brzezinski
  • Kyoko Furuita
  • Chojiro Kojima
  • Sneha Shah
  • Evgenii L. KovriginEmail author
  • Tamo FukamizoEmail author


Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases.


Line shape analysis NMR Chitosanase Chitosan Induced fit Exchange regime HSQC titration IDAP TITAN 



This work was supported by “Strategic Project to Support the Formation of Research Bases at Private Universities: Matching Fund Subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2011–2015 (S1101035) and partially supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science from Japan Agency for Medical Research and development (AMED) to TF. Work at Université de Sherbrooke was sustained by a Discovery Grant from the Natural Science and Engineering Research Council of Canada to RB. ELK acknowledges Committee on Research (COR) Summer Faculty Fellowship 2012 from Marquette University. SS was supported by a Research Fellowship for Young Scientists from Japan Society for the Promotion of Science (25-3639).

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Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Shoko Shinya
    • 1
    • 3
  • Mariana G. Ghinet
    • 2
    • 5
  • Ryszard Brzezinski
    • 2
  • Kyoko Furuita
    • 3
  • Chojiro Kojima
    • 3
  • Sneha Shah
    • 4
  • Evgenii L. Kovrigin
    • 4
    Email author
  • Tamo Fukamizo
    • 1
    Email author
  1. 1.Department of Advanced BioscienceKindai UniversityNaraJapan
  2. 2.Département de Biologie, Faculté des SciencesUniversité de SherbrookeSherbrookeCanada
  3. 3.Institute for Protein ResearchOsaka UniversitySuitaJapan
  4. 4.Department of ChemistryMarquette UniversityMilwaukeeUSA
  5. 5.Département de Pharmacologie, Faculté de Médecine et des Sciences de la SantéUniversité de SherbrookeSherbrookeCanada

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