Last modified 19th May '95 © Birkbeck College 1995

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The function of lysozyme is to hydrolyze the ß(1-4) glycosidic bond between residues of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in certain polysaccharides.

Here is a diagram of the structure of these sugars. Substrates of lysozyme include:

Here is a diagram of the backbone structure of bacterial cell wall polysaccharide. Oligopeptide side chains may be bonded to the sugar residues. Examine this NAM-NAG-NAM trisaccharide.

Structure of lysozyme

Hen egg white lysozyme is a single chain of 129 residues. It has an alpha+beta fold, consisting of five to seven alpha helices and a three-stranded antiparallel beta sheet. The enzyme is approximately ellipsoidal in shape, with a large cleft in one side forming the active site.

More diagrams:icon icon . . see the cleft.

Here is the pdb structure file. Because the rate of conversion of substrate into product is much higher than the rate of diffusion of substrate into the enzyme molecules in a crystal, obtaining an X-ray structure of the enzyme with bound substrate is highly problematic.

Structure of lysozyme complexed with a competitive inhibitor

The trisaccharide NAG-NAG-NAG (tri-NAG) binds strongly to the active site. Its rate of hydrolysis is negligible. Crystallization of tri-NAG bound to lysozyme indicated the position of the active site.

Here are diagrams of space-filling models of the complex (with tri-NAG represented by space-filling model, and by liquorice model), in which the residues of the active site are highlighted.

Tri-NAG occupies about half of the cleft. The following hydrogen bonding interactions are apparent between the active site and the inhibitor:

Additionally, the second residue (B) interacts closely with the indole ring of Trp 62, making van der Waals contacts.

Examine these interactions in the crystal structure.

Binding of hexa-NAG

The crystal structure indicates that three more NAG residues are required to fill the entire cleft; model-building suggested the manner of binding of a complete hexa-NAG oligosaccharide. The site is therefore considered to consist of six subsites, each of which binds one sugar residue (labelled A-F).

Note that there is a marked increase in the rate of hydrolysis of penta-NAG compared with tetra-NAG or tri-NAG (both extremely low- the rate for tri-NAG is 4000 times lower than for penta-NAG); there is a further eight-fold increase if the number of residues in increased from five to six. However the rate is no higher for seven- or eight-residue NAG oligomers compared to the hexamer.

When the hexa-NAG substrate is bound to the active site, the fourth NAG residue must be distorted in order to fit: whereas the residues usually have the non-planar 'chair' conformation (in which no two consecutive atoms of the ring are in the same plane- see NAM-NAG-NAM trisaccharide) this fourth residue has a 'sofa' conformation when bound.

Hydrolysis of the cell wall polysaccharide

Neither the bond between residues A and B nor the B-C bond can be the one which is hydrolyzed by the enzyme, as tri-NAG is stable. Model-building also indicates that a NAM residue cannot fit into subsite C, because this sugar has a lactyl side chain. Therefore sites A, C and E must be occupied by NAG residues, and B, D and F by NAM, rather than the other way round. Since only NAM-NAG glycosidic bonds (i.e. between C-1 of NAM and C-4 of NAG) are cleaved, and not NAG-NAM bonds, bonds A-B, C-D and E-F are excluded as the candidate for hydrolysis. Therefore the cleavage must occur between residues D and E.

Studies involving labelling with water containing oxygen-18 indicated that the hydrolyzed bond is that between C-1 of NAM (residue D) and the oxygen of the glycosidic bond (rather than that linking the O with C-4 of NAG residue E). This allowed a search for catalytic groups in a very localized area of the protein structure. It was therefore deduced that Glu 35 (which has a non-polar environment, and is likely to be non-ionized at the optimum pH (5) for enzyme activity) and Asp 52 (ionized, in a polar environment) are the principal catalytic residues.

T4 lysozyme

Lyzome from the bacteriophage T4 is somewhat larger than that from hen egg white (164 residues) and contains several more alpha helices. T4 lysozyme can only hydrolyze substrates which have peptide side chains bonded to the polysaccharide backbone. The cell wall polysaccharide of Escherichia coli has a peptide chain covalently bonded to the lactyl chain of NAM. The sequence of this peptide chain is
L-Ala, D-Glu, diaminopimelic acid (DAP), D-Ala
The laevo Alanine is bonded to NAM. Here is the complete disaccharide with bonded peptide

Chitin, which has no such peptide constituent, cannot be hydrolyzed by T4 lysozyme.

A mutant T4 lysozyme has been crystallized in which Thr 26 in the active site cleft is replaced by Glu (Kuroki et al., 1993). This mutant is still able to cleave the E. coli polysaccharide, but the disaccharide product remains covalently bound (in subsites C and D) to the enzyme at Glu 26 (which is bonded to C-1 of NAM). The peptide chain lies across the surface of the protein, approximately inbetween two of the helices as highlighted in these three diagrams. Here is the structure file- the NAM residue is named "AMU" and DAP "API".


Enzyme Databases

Lysozymes in


Here is the index to the Protein Science Kinemages on Lysozymes.

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J. Walshaw