title: A novel transition-state analogue for lysozyme, 4-O-β-tri-N-acetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate.
abstract: 4-O-β-Di-N-acetylchitobiosyl moranoline (2) and 4-O-β-tri-N-acetylchitotriosyl moranoline (3) were produced by lysozyme-mediated transglycosylation from the substrates tetra-N-acetylchitotetraose, (GlcNAc)4, and moranoline, and the binding modes of 2 and 3 to hen egg white lysozyme (HEWL) was examined by inhibition kinetics, isothermal titration calorimetry (ITC), and x-ray crystallography. Compounds 2 and 3 specifically bound to HEWL, acting as competitive inhibitors with Ki values of 2.01 × 10(-5) and 1.84 × 10(-6) m, respectively. From ITC analysis, the binding of 3 was found to be driven by favorable enthalpy change (ΔHr°), which is similar to those obtained for 2 and (GlcNAc)4. However, the entropy loss (-TΔSr°) for the binding of 3 was smaller than those of 2 and (GlcNAc)4. Thus the binding of 3 was found to be more favorable than those of the others. Judging from the Kd value of 3 (760 nm), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. X-ray crystal structure of HEWL in a complex with 3 showed that compound 3 binds to subsites -4 to -1 and the moranoline moiety adopts an undistorted (4)C1 chair conformation almost overlapping with the -1 sugar covalently bound to Asp-52 of HEWL (Vocadlo, Davies, G. J., Laine, R., and Withers, S. G. (2001) Nature 412, 835-838). From these results, we concluded that compound 3 serves as a transition-state analogue for lysozyme providing additional evidence supporting the covalent glycosyl-enzyme intermediate in the catalytic reaction.
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ogata
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Umemoto
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Numata
authors.forename: Akari
authors.initials: A
authors.lastname: Suzuki
authors.forename: Taichi
authors.initials: T
authors.lastname: Usui
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives* / Q000737:chemistry*; D000818:Animals; D002384:Catalysis; D002645:Chickens; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry*; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D004791:Enzyme Inhibitors; D017485:1-Deoxynojirimycin; D009113:Muramidase