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

Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimens ional structure determined by X-ray diffraction techniques(1). A catalytic mechanism, featuring a long-lived oxo-carbenium-ion intermediate, was propo sed on the basis of model-building studies(2). The `Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta -glycosidas es that cleave glycosidic linkages with net retention of configuration of t he anomeric centre. Studies with other retaining beta -glycosidases, howeve r, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postu lated(3). Here we show, in three different cases using electrospray ionizat ion mass spectrometry, a catalytically competent covalent glycosyl-enzyme i ntermediate during the catalytic cycle of HEWL. We also show the three-dime nsional structure of this intermediate as determined by Xray diffraction. W e formulate a general catalytic mechanism for all retaining beta -glycosida ses that includes substrate distortion, formation of a covalent intermediat e, and the electrophilic migration of C1 along the reaction coordinate.