title: NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase.
abstract: 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.
authors.affiliation: Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara, 631-8505, Japan.|Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Shinya
authors.affiliation: Département de Biologie, Faculté des Sciences, Université de Sherbrooke, 2500, boul. de l'Université, Sherbrooke, QC, J1K 2R1, Canada.|Département de Pharmacologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, J1H 5N4, Canada.
authors.forename: Mariana G
authors.initials: MG
authors.lastname: Ghinet
authors.affiliation: Département de Biologie, Faculté des Sciences, Université de Sherbrooke, 2500, boul. de l'Université, Sherbrooke, QC, J1K 2R1, Canada.
authors.forename: Ryszard
authors.initials: R
authors.lastname: Brzezinski
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Furuita
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Chojiro
authors.initials: C
authors.lastname: Kojima
authors.affiliation: Department of Chemistry, Marquette University, Milwaukee, WI, 53201, USA.
authors.forename: Sneha
authors.initials: S
authors.lastname: Shah
authors.affiliation: Department of Chemistry, Marquette University, Milwaukee, WI, 53201, USA. evgueni.kovriguine@marquette.edu.
authors.forename: Evgenii L
authors.initials: EL
authors.lastname: Kovrigin
authors.affiliation: Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara, 631-8505, Japan. fukamizo@nara.kindai.ac.jp.
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D048271:Chitosan / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase
keywords: Chitosan; Chitosanase; Exchange regime; HSQC titration; IDAP; Induced fit; Line shape analysis; NMR; TITAN