1. Protein Engineering Network of Centres of Excellence and the Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada
2. Department of Chemistry, Structural Biology Laboratory, University of York, Heslington, York YO10 5DD, UK
3. Departments of Biology and Chemistry, Louisiana State University, Baton Rouge, Louisiana 70808, USA

Correspondence to: Stephen G. Withers¹ Correspondence and requests for materials should be addressed to S.G.W. (e-mail: Email: withers@chem.ubc.ca). The structure for the HEWL covalent intermediate has been deposited in the Protein DataBank under accession code 1h6m.

Abstract

Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques¹. A catalytic mechanism, featuring a long-lived oxocarbenium-ion intermediate, was proposed on the basis of model-building studies². The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of -glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining -glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated³. 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 -glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.