From Proteopedia
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Lysozyme was the first enzyme whose x-ray structure was determined. The folllowing scene, shows this structure containing a trisaccharide of N-acetylglucosamine (NAG) bound to the active site. David Phillips, who determined the structure in 1965, saw that the active site was large enough to fit three more saccharide units and his group built a model extending the trisaccharide to a hexasaccharide
fit into the active site. Alternately click on and to turn the model portion of the hexasaccharide on and off.
The interesting thing about the model-building experiment was that the only way that the hexasaccharide would fit into the active site was for the 4th saccharide to be strained into a . This conformation is what would be necessary for the formation of an oxocarbenium ion (oxionium ion). When the model was studied, was found to be in an ideal location to act as a general acid catalyst, protonating bridging oxygen between the 4th and 5th saccharide units. was too far away in the static lysozyme structure to have formed a covalent bond with C1 of the half-chair hexasaccharide 4, so Phillips proposed that it acted as an electrostatic stabilizer of the oxonium ion.
Then, in 2001, Stephen Withers published , in which Glu 35 was mutated to Gln to remove general acid catalysis and the substrate contained NAG-2-fluoro-glucosyl fluoride (NAG2FGlcF). The fluoro group does not require acid catalysis to be a good leaving group, and the remaining saccharide, in the absence of the acid necessary to catalyse the second step of the reaction, was demonstrated to . Note in this of half chair model with 1HEW (greens) on covalent intermediate in 1H6M (blues) the motion of Asp 52 and C1 of the sugar ring in going from the model to the covalent intermediate.
