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

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 β-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining β-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 β-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.

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Figure 1: Two possible catalytic mechanisms for HEWL.
Figure 2: Transform of the ESI-MS mass spectra of HEWL and HEWL complexes.
Figure 3: Structure of the covalent intermediate in the HEWL reaction.
Figure 4: Comparison of hexopyranose covalent intermediates.

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Acknowledgements

We thank N. Strynadka, Y. Luo and S. Mosimann for access to the X-ray source and computer time; J. Kirsch for supplying the DNA containing the HEWL(E35Q) clone; G. Dodson, M. Sinnott and J. Voet for critical reading of the manuscript; S. He for expert technical assistance; and H. Brummer III and S. Williams for discussions. This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Protein Engineering Network of Centres of Excellence of Canada. The York Structural Biology Laboratory is supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust. We thank the Peter Wall Institute for Advanced Studies for supporting G.J.D. under the catalytic visitor programme to the University of British Columbia. D.J.V. is a NSERC predoctoral scholar; G.J.D. is a Royal Society University Research Fellow.

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Correspondence to Stephen G. Withers.

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Vocadlo, D., Davies, G., Laine, R. et al. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature 412, 835–838 (2001). https://doi.org/10.1038/35090602

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