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.
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
- chitin, which is a polymer of NAG linked by ß(1-4) glycosidic
bonds. Chitin is found in crustacean shell tissue
- the polysaccharide component of the cell walls of certain bacteria; the
principal function of lysozyme is as an antibacterial agent. This polysaccharide
is composed of alternating residues of NAG and NAM. Only the glycosidic bonds
between C-1 of NAM and C-4 of NAG are hydrolyzed.
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.
. . see the cleft.
Here is the pdb structure
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.
- The side chain of Asp 101 interacts with both the first (residue A, see below)
and second (B) NAG residues
- Trp 62 and Trp 63 side chains hydrogen bond to the third NAG residue (C)
- The main chain of residues 59 and 107 also interact with the third NAG (C)
Examine these interactions in the
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
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.
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
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
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
diagrams. Here is the structure
file- the NAM residue is named "AMU" and DAP "API".
- Anderson, W.F., Grutter, M.G, Remington, S.J. and Matthews, B.W. (1981)
Crystallographic determination of the mode of binding of oligosaccharides
to bacteriophage-T4 lysozyme - implications for the mechanism of catalysis
J. Mol. Biol. 147, 523-543
- Cheetham, J.C., Artymiuk, P.J. and Phillips, D.C. (1992) Refinement of an
Enzyme Complex with Inhibitor Bound at Partial Occupancy. Hen Egg-white
Lysozyme and Tri-N-Acetylchitotriose at 1.75Å Resolution J. Mol. Biol.
- Kelly, J.A., Sielecki, A.R., Sykes, B.D., James, M.N.G. and Phillips, D.C.
(1979) Nature 282, 875
- Kuroki, R., Weaver, L.H. and Matthews, B.W. (1993) A Covalent Enzyme-substrate
Intermediate with Saccharide Distortion in a Mutant T4 Lysozyme Science
- Phillips, D.C. (1986) The three-dimensional Structure of an Enzyme Molecule
Sci. Amer. 215(5), 78-90
- Stryer, L. (1981) Biochemistry, W.H. Freeman & Co., New York, Chapter 7
Here is the index to the Protein Science
Kinemages on Lysozymes.
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