title: Thermodynamic analysis of allosamidin binding to a family 18 chitinase.
abstract: Inhibition of family 18 chitinases is emerging as a target for pest and fungal control as well as asthma and inflammatory therapy. One of the best known inhibitors for these enzymes is allosamidin, a natural product. While interactions of this compound with family 18 chitinases have been studied in much detail by X-ray crystallography and standard enzymology, details of the driving forces behind its tight binding remain unknown. We have studied the thermodynamics of allosamidin binding to chitinase B (ChiB), a family 18 chitinase from Serratia marcescens, using isothermal titration calorimetry. At pH 6.0, Kd is 0.16 +/- 0.04 microM, and the binding reaction is entropically driven (DeltaSr = 44 cal/K mol) with an enthalpic penalty (DeltaHr = 3.8 +/- 0.2 kcal/mol). Dissection of the entropic term shows that a favorable conformational change in the allosamidin-ChiB complex (DeltaSconf = 37 cal/K mol) is the main contributor to the reaction. At pH 8.5, Kd decreases to 0.03 muM and the binding reaction is less entropically favorable (DeltaSr = 30 cal/K mol). While the solvation entropy change (DeltaSsolv) increases from 15 cal/K mol at pH 6.0 to 46 cal/K mol at pH 8.5, DeltaSconf becomes small and negative (-8 cal/K mol) because of an enthalpy-entropy compensation. Analyses of proton transfer showed that at pH 6.0 binding of allosamidin requires deprotonation of the Asp142-Glu144 catalytic diad. At pH 8.5, the 142-144 diad is ionized in the native enzyme, relieving the deprotonation penalty of binding and explaining why binding becomes enthalpically favorable (DeltaHr = -1.2 +/- 0.2 kcal/mol).
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N - 1432 As, Norway.
authors.forename: F Henning
authors.initials: FH
authors.lastname: Cederkvist
authors.forename: Silje F
authors.initials: SF
authors.lastname: Saua
authors.forename: Vigdis
authors.initials: V
authors.lastname: Karlsen
authors.forename: Shohei
authors.initials: S
authors.lastname: Sakuda
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
authors.forename: Morten
authors.initials: M
authors.lastname: Sørlie
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002688:Chitinases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D037102:Lectins / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D012706:Serratia marcescens / Q000201:enzymology; D013816:Thermodynamics; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D037102:Lectins; D014312:Trisaccharides; C054851:allosamidin; D002688:Chitinases; D000117:Acetylglucosamine