Lysozyme is an enzyme that catalyzes the breakdown of bacterial cell walls. It was first discovered in 1922 by Alexander Fleming. The enzyme hydrolyzes beta linkages between N-acetylglucosamine and N-acetylmuramic acid in peptidoglycan. Lysozyme has antibacterial properties and is found naturally in human tears, saliva, and egg whites. It has important applications in protein purification and nucleic acid preparation. The enzyme has a compact globular structure and contains four disulfide bonds that provide stability. Its active site contains the residues glutamic acid 35 and aspartic acid 52, which work together to catalyze the hydrolysis reaction.

1. Submitted by;
Upasna
Thisarani
Chanaka
Sumudu
Presented to;
Mr. Jafar Hasbullah
LYSOZYME
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2. Introduction
History
Occurrence
Functions
Properties
Applications
Structure
Catalysis
Role of the Active Site
Substrate, Inhibitors & Activators
Biomedical Importance
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3. Introduction
 Lysozyme: is 129 amino acid residues enzyme .
 It is a basic bacteriolytic Protein that hydrolyzes peptidoglycan, glycosidic bonds and are widely
distributed in nature.
 (EC 3.2.1.17), hydrolase which catalyzes hydrolysis of 1,4-beta-linkages between N-
acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan and between N-acetyl-
D-glucosamine residues in chitodextrins.
 Molecular weight of Lysozyme is an approximately 14.7 kDa .
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4. Protein Accession Number: P00698
CATH Classification (v. 3.2.0):
Class: Mainly Alpha
Architecture: Orthogonal Bundle
Topology: Lysozyme
Optimal pH: 6.0-9.0
Isoelectric Point: 9.32
Extinction Coefficient:
38,940 cm-1 M-1
E1%,280 = 27.21
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6. History
 Laschtschenko first discovered Lysozyme in 1909,
when he first observed the antibacterial property of
hen egg whites.
 Lysozyme was discovered in 1922 by Alexander
Fleming on a remarkable becteriolytic element found
in tissues and secretion.
 In 1965 the structure of Lysozyme was solved by X-
Ray analysis with 2 angstrom resolution by David
Chilton Phillips.
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7. Occurrence
An enzyme found naturally in;
 Hen Egg White
 Human Tears
 Saliva (as well as other body fluids)
 Many Animal Fluids
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8. Functions
 As an antibacterial agent by catalyzing
the hydrolysis of specific glycosidic
linkages in peptidoglycan and chitin,
breaking down some bacterial cell walls.
 hydrolyze the ß(1-4) glycosidic bond between
residues of N-acetylmuramic acid (NAM) and
N-acetylglucosamine (NAG) in certain
polysaccharides.
 Acts as a mild antiseptic.
 As a model protein for studying on structure and
function of protein.
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9. Properties
 Lysozyme has the characters of a ferment. The rapidity of its
action increases up to 60° C, but at temperatures over 65° C.
it is destroyed more or less rapidly.
 It acts best in a neutral medium.
 Peptic or tryptic digestion does not destroy Lysozyme.
 Stability—When kept dry, Lysozyme can be preserved for a
long time. It was noted that commercial dried egg albumen
was very rich in Lysozyme .
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11. Applications
 Nucleic acid preparation.
 Protein purification from inclusion bodies.
 Plasmid preparation (to break down membranes and
cell wall).
 Hydrolysis of chitin.
 Hydrolysis of bacterial cell walls.
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16. Structure
 Lysozyme is a compact protein of 129 amino acids which folds into a
compact globular structure.
 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.
 It comprises of 2 domains joined by a long Alpha helix between
which lies the active site for antimicrobial activity;
 N-terminal domain( residues 40-88) has some helices and Beta
parallel sheets.
 The Second domain (1-39 and 89-129) has mostly Alpha helical
structure.
 4 Sulphide bonds in locations between Cys 6-Cys127,Cys30-
Cys115,Cys 64-Cys 80 and Cys 76-Cys 94 lend stability and unusual
compaction.
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17. 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:
 The side chain of Asp 101 interacts with both the first and second NAG
residues.
 Trp 62 and Trp 63 side chains hydrogen bond to the third NAG residue.
 The main chain of residues 59 and 107 also interact with the third NAG.
 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..
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18. 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.
This fourth residue has a 'sofa' conformation when bound.
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19. 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.
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20. 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 isL-Ala, D-Glu, diaminopimelic
acid (DAP), D-AlaThe laevo Alanine is bonded to NAM.
 Here is the complete disaccharide with bond 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 .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 in
between two of the helices.
 Here is the structure file-the NAM residue is named "AMU" and DAP "API".
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21. Catalysis
 For catalysis to occur, (NAG-NAM)3 binds to the active site with each
sugar in the chair conformation except the fourth which is distorted to
a half chair form, which labilizes the glycosidic link between the 4th
and 5th sugars.
 if the sugars that fit into the binding site are labeled A-F, then
because of the bulky lactyl substituent on the NAM, residues C and E
can not be NAM, which suggests that B, D and F must be NAM
residues.
 Cleavage occurs between residues D and E. Catalysis by the
enzyme involves Glu 35 and Asp 52 which are in the active site.
 Asp 52 is surrounded by polar groups but Glu 35 is in a hydrophobic
environment.
 This should increase the apparent pKa of Glu 35, making it less
likely to donate a proton and acquire a negative charge at low pH
values, making it a better general acid at higher pH values. The
general mechanism appears to involve.
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binding of a hexasaccharide unit of the peptidoglycan with concomitant distortion
of the D NAM.
protonation of the sessile acetal O by the general acid Glu 35 (with the elevated
pKa), which facilitates cleavage of the glycosidic link and formation of the resonant
stabilized oxonium ion.
Asp 52 stabilizes the positive oxonium through electrostatic catalysis.
The distorted half-chair form of the D NAM stabilizes the oxonium which requires
co-planarity of the substituents attached to the sp2 hybridized carbon of the
carbocation resonant form (much like we saw with the planar peptide bond).
water attacks the stablized carbocation, forming the hemiacetal with release of
the extra proton from water to the deprotonated Glu 35 reforming the general acid
catalysis.

23. Mechanism of Acetyl
Cleavage
 Binding and distortion of the D substituent of the substrate (to the
half chair form as shown Below) occurs before catalysis.
 Since this distortion helps stabilize the oxonium ion intermediate, it
presumably stabilizes the transition state as well.
 Hence this enzyme appears to bind the transition state more tightly
than the free, undistorted substrate, which is yet another method of
catalysis.
 pH studies show that side chains with pKa's of 3.5 and 6.3 are
required for activity.
 These presumably correspond to Asp 52 and Glu 35, respectively.
 If the carboxy groups of lysozyme are chemically modified in the
presence of a competitive inhibitor of the enzyme, the only protected
carboxy groups are Asp 52 and Glu 35.
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A recent paper suggests an alternative mechanism to the Phillips mechanism
above.
 In this case, Asp 52 acts as a nucleophilic catalysis and forms a covalent bond
with NAM, expelling a NAG leaving group with Glu 35 acting as a general acid.
 This alternative mechanism also is consistent with other b-glycosidic bond
cleavage enzyme.
 Substrate distortion is also important in this alternative mechanism.

25. Role of the Active Site
 The X –Ray Crystallography structure of Lysozyme has been determined in the
presence of a non-hydrolyzable substrate analog. This analog binds tightly in the
enzyme's active Site to form the ES complex, but ES cannot be efficiently converted
to EP. It would not be possible to determine the X-ray structure in the presence of the
true substrate, because it would be cleaved during crystal growth and structure
determination.

The active site consists of a crevice or depression that runs across the surface of the
enzyme. Look at the many enzymes contacts between the substrate and enzyme
active site that enables the ES complex to form. There are 6 subsites within the
crevice, each of which is where hydrogen bonding contacts with the enzymes are
made. In site D, the conformation of the sugar is distorted in order to make the
necessary hydrogen bonding contacts. This distortion raises the energy of the ground
state, bringing the substrate closer to the transition state for hydrolysis.
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26. Active Site Residues
 Glutamic acid (E53)
 Aspartic acid (D70)
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28. Substrate
 The beta (1-4) glycosidic bond between N-
acetylglucosamine sugar (NAG) and N-
acetylmuramic acid sugar (NAM) to be hydrolysed
during the Lysozyme reaction are circled.
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Inhibitors
SDS
Alcohols
N-acetyle-D-glucosamine
Oxidizing agents
Activators
EDTA

30. Biomedical Importance
 Important defence molecule of fish innate immune system.
 Lysozyme is part of the innate immune system.
 Reduced lysozyme levels have been associated with broncho
pulmonary dysplasia in newborns.
 Since lysozyme is a natural form of protection from gram-positive
pathogens like Bacillus and Streptococcus
 In certain cancers (especially myelomonocytic leukaemia) excessive
production of lysozyme by cancer cells can lead to toxic levels of
lysozyme in the blood.
 High lysozyme blood levels can lead to kidney failure and low blood
potassium.
 Significance for the bactericidal effects of milk, its changes in mastitis
and the resulting possibility of its introduction in diagnostic work, and
the therapeutical use of milk rich in lysozyme. 30

31. Conclusion
 lysozyme is a widely distributed antibacterial ferment which is probably inherent in all
animal cells and constitutes a primary method of destroying bacteria.
 While acting most strikingly on non-pathogenic bacteria yet can, when allowed to act
in the full strength in which it occurs in some parts of the body, attack pathogenic
organisms.
 That it is very easy to make bacteria relatively resistant to lysozyme, so that any
pathogenic microbe isolated from the body where it has been growing in the presence
of a non-lethal concentration of lysozyme must have acquired increased resistance to
the ferment.
 There are some differences in the lysozyme of different tissues and in different
animals whereby bacteria are susceptible to different lysozymes in varying degrees.
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32. Reference
 slideshare.net
 onlinelibrary.wiley.com
 ncbi.nlm.nih.gov
 lysozyme.co.uk
 novapublishers.com
 On a Remarkable Bacteriolytic Element
found in Tissues and Secretions.
By ALEXANDER FLEMING, M.B.,F.R.C.S.
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