Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate.
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.
Vocadlo
D J
DJ
Protein Engineering Network of Centres of Excellence and the Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
Davies
G J
GJ
Laine
R
R
Withers
S G
SG
D000818:Animals; D002384:Catalysis; D002645:Chickens; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization
C421864:hen egg lysozyme; D009113:Muramidase