Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

Nature. 2001 Aug 23;412(6849):835-8. doi: 10.1038/35090602.

Abstract

Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxocarbenium-ion intermediate, was proposed on the basis of model-building studies. The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by X-ray diffraction. We formulate a general catalytic mechanism for all retaining beta-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Catalysis
  • Chickens
  • Crystallography, X-Ray
  • Models, Molecular
  • Muramidase / chemistry
  • Muramidase / genetics
  • Muramidase / metabolism*
  • Mutation
  • Protein Conformation
  • Spectrometry, Mass, Electrospray Ionization

Substances

  • hen egg lysozyme
  • Muramidase

Associated data

  • PDB/1H6M