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

Makoto Ogata, Naoyuki Umemoto, Takayuki Ohnuma, Tomoyuki Numata, Akari Suzuki, Taichi Usui, Tamo Fukamizo

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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 Ki 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 (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. Thusthe binding of 3 was found to bemorefavorable 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 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.

Original languageEnglish
Pages (from-to)6072-6082
Number of pages11
JournalJournal of Biological Chemistry
Volume288
Issue number9
DOIs
Publication statusPublished - Mar 1 2013
Externally publishedYes

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1-Deoxynojirimycin
Muramidase
Egg White
Enzymes
X-Rays
Calorimetry
Crystallography
Entropy
X rays
Titration
Sugars
Conformations
Enthalpy
Crystal structure
Kinetics
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A novel transition-state analogue for lysozyme, 4-O-β-Tri-N- acetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate. / Ogata, Makoto; Umemoto, Naoyuki; Ohnuma, Takayuki; Numata, Tomoyuki; Suzuki, Akari; Usui, Taichi; Fukamizo, Tamo.

In: Journal of Biological Chemistry, Vol. 288, No. 9, 01.03.2013, p. 6072-6082.

Research output: Contribution to journalArticle

Ogata, Makoto ; Umemoto, Naoyuki ; Ohnuma, Takayuki ; Numata, Tomoyuki ; Suzuki, Akari ; Usui, Taichi ; Fukamizo, Tamo. / A novel transition-state analogue for lysozyme, 4-O-β-Tri-N- acetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate. In: Journal of Biological Chemistry. 2013 ; Vol. 288, No. 9. pp. 6072-6082.
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abstract = "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 Ki 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 (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. Thusthe binding of 3 was found to bemorefavorable 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 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.",
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