1. 1. Submitted by; Upasna Thisarani Chanaka Sumudu Presented to; Mr. Jafar Hasbullah LYSOZYME 1
2. 2. Introduction History Occurrence Functions Properties Applications Structure Catalysis Role of the Active Site Substrate, Inhibitors & Activators Biomedical Importance 2
3. 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 . 3
4. 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 4
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6. 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. 6
7. 7. Occurrence An enzyme found naturally in; Hen Egg White Human Tears Saliva (as well as other body fluids) Many Animal Fluids 7
8. 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 (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. 8
9. 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. StabilityWhen kept dry, Lysozyme can be preserved for a long time. It was noted that commercial dried egg albumen was very rich in Lysozyme . 9
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11. 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. 11
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16. 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. 16
17. 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.. 17
18. 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. 18
19. 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. 19
20. 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". 20
21. 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. 21
22. 22. 22 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. 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. 23
24. 24. 24 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. 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. 25
26. 26. Active Site Residues Glutamic acid (E53) Aspartic acid (D70) 26
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28. 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. 28
29. 29. 29 Inhibitors SDS Alcohols N-acetyle-D-glucosamine Oxidizing agents Activators EDTA
30. 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. 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. 31
32. 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. 32
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