Tutorial developed by Ross Feldberg, Dept. of Biology, Tufts University
Background on Lysozyme
Lysozyme is an enzyme found in tears, nasal secretions and the white of avian eggs which hydrolyzes the polysaccharides found in many bacterial cell walls. As such, it has mild antibacterial action and indeed was one of the first antibiotics studied by Sir Fleming, the discover of penicillin. Hen egg white lysozyme has been studied in great detail. Because the enzyme is a very effective catalyst, it has been impossible to obtain a stable crystal between enzyme and substrate. However, it has been possible to obtain crystals of the enzyme complexed to trisaccharide inihibitors. The structure shown here contains a trisaccharide of N-acetylglucosamine (NAG3) and is from the pdb file 1HEW.
Lysozyme is a compact protein of 129 amino acids which folds into a compact globular structure. Note as the protein rotates that there is a rather deep cleft in the protein surface into which six carbohydrates can bind. It is possible to crystallize the protein in the presence of a trisaccharide which is a competitive inhibitor of the substrate. That trisaccharide, (NAG)3 is shown here in blue.
Lysozyme has five helical regions. Three are standard alpha helices but one (residues 109- 115) is closer to the pi helix in character while two (80-84 and 120-124) are intermediate in structure between the 3-10 helix and the alpha helix. There are also five regions of beta sheet and a large amount of random coil and beta turns. Although this representation shows secondary structure, the openness of it makes it impossible to identify the cleft.
This button selects a series of residues in the protein that have been identified as taking part in binding of the oligosaccharide substrate. Although these residues make up the surface of a deep groove in the protein, they can be quite far apart in the primary structure. The residues selected are, in order: asp101 (partially buried); trp62, trp63, asn59, ala107; val109 and gln57. The next residues (asp52 and glu35) are colored magenta since they are particularly important in the catalytic mechanism; gln57, asn44 phe34 and arg114. Finally, the trisaccharide inhibitor is shown bound to the initial residues.
Two carboxylic acid residues play an important role in catalyzing the hydrolysis of the oligosaccharide substrate. A key factor (not shown here) is that the protein distorts the sugar at the fourth subsite along the protein surface into a half chair conformation which is similar to the transition state. In addition, glu35 transfers a proton to the O1 position while the negative charge on asp52 stabilizes the positively charged oxonium ion intermediate. However, for glu35 to be protonated at neutral pH, its dissociation must have been suppressed. This is accomplished by placing it in a very hydrophobic environment. In this view, asp 52 and glu35 are initially shown and then hydrophobic groups within 4.5A of each residue are selected and colored green. It is clear that the glu35 carboxylic acid side chain is surrounded by hydrophobic groups while that of asp52 is in a polar environment.
This tutorial was developed by Ross S. Feldberg (Dept. of Biology,
Tufts University) with
help from a Teaching with Technology grant from the Center for Teaching Excellence at Tufts.
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