A novel transition-state analogue for lysozyme, 4-O-β-Tri-N- acetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate

4-O-β-Di-N-acetylchitobiosyl moranoline (2) and 4-O-β-tri- Nacetylchitotriosyl 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 K_i values of 2.01 × 10^-5 and 1.84 × 10^-6 M, respectively. From IT Canalysis, the binding of 3 was found to be driven by favorable enthalpy change (ΔH_r°), which is similar to those obtained for 2 and (GlcNAc)₄. However, the entropy loss (-TΔS_r°) for the binding of 3 was smaller than those of 2 and (GlcNAc)₄. Thusthe binding of 3 was found to bemorefavorable than those of the others. Judging from the K_d 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 HEWLin a complex with 3 showed that compound 3 binds to subsites -4 to -1 and the moranoline moiety adopts an undistorted ⁴C₁ chair conformation almost overlapping with the -1 sugar covalentlyboundtoAsp-52ofHEWL(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.