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Not to be confused with Lysosome or Lysosomal enzymes.
Lysozyme
Lysozyme crystals stained with methylene blue.
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EC no.3.2.1.17
CAS no.9001-63-2
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Lysozymes, also known as muramidase or N-acetylmuramide glycanhydrolase, are glycoside hydrolases which damage bacterial cell walls by catalyzing the hydrolysis of peptidoglycan, which is the major component of gram-positive bacterial cell walls. Lysozymes catalyze hydrolysis of the β-(1,4) linkages in the bacteria by compromising the integrity of its cell wall.

Lysozyme is abundant in a number of secretions, such as tears, saliva, human milk, and mucus. It is also present in cytoplasmic granules of the macrophages and the polymorphonuclear neutrophils (PMNs). Large amounts of lysozyme can be found in egg white. C-type lysozymes are closely related to alpha-lactalbumin in sequence and structure, making them part of the same family. In humans, the lysozyme enzyme is encoded by the LYZ gene.[1][2]

Lysozyme is thermal stable, whose melting point can reach up to 72 ℃ at pH 5.0.[3] But in human milk it loses activity very fast at that temparature.[4] Its isoelectric point is 11.35. In a large range of pH (6-9) lysozyme can survive.[5]

Function and mechanism

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The enzyme functions by attacking, hydrolyzing, and breaking glycosidic bonds in peptidoglycans. The enzyme can also break glycosidic bonds in chitin, although not as effective as true chitinases[6]. The ability to break down both oligosaccharides suggests a similar mechanism between the breakdown of the two molecules.

Overview of the reaction

Lysozymes active site binds the peptidoglycan molecule in the prominent cleft between its two domains. It attacks peptidoglyans (found in the cell walls of bacteria, especially Gram-positive bacteria), its natural substrate, between N-acetylmuramic acid(NAM) and the fourth carbon atom of N-acetylglucosamine(NAG). Shorter saccharides like tetrasaccharide have also shown to be viable substrates but via an intermediate with a longer chain[7]. Chitin has also been show to be a viable lysozyme substrate. Artificial substrates have also been developed and used in lysozyme.[8]

HEWL E35Q Mutant where D35 is bound to a non-native substrate PDB: 1H6M
HEWL active site before it binds its substrate PDB: 1DPX

Mechanism

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Phillips Mechanism

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The Phillips Mechanism proposed that the enzyme's catalytic power came from both steric strain on the bound substrate and electrostatic stabilization of the oxo-carbenium intermediate. From x-ray crystallography data, Phillips proposed the lysozyme's active site binds to a hexasaccharide. The lysozyme distorts the fourth sugar in hexasaccharide into a half-chair conformation. In this stressed state, the glycosidic bond is more easily broken[9]. An ionic intermediate containing an oxo-carbenium is created as a result of the the glycosidic bond breaking.[10] Thus distortion causing the substrate molecule to adopt a strained conformation similar to that of the transition state will lower the energy barrier of the reaction[11].

This oxo-carbonium intermediate was also proposed to be electrostatically stabilized by Arieh Warshel in 1978. Residues in the active site, such as aspartate and glutamate are able to stabilize this intermediate. The electrostatic stabilization argument was based on comparison to bulk water, the reorientation of water dipoles can cancel out the stablizing energy of charge interaction. In Warshel's model, the enzyme acts as a super-sovlent, which fixes the orientation of ion pairs and provides super-solvation (very good stablization of ion pairs), and especially lower the energy when to ions are close to each other.[12]

The rate-determining step(RDS) in this mechanism is related to formation of the oxo-carbenium intermediate. There were some contadictory results to indicate the exact RDS. By tracing the formation of product (p-nitrophenol), it was discovered that the RDS can change over different temparatures, which was a reason for those contadictory results. At a higher temparature the RDS is formation of glycosyl enzyme intermediate and at a lower temparature the break down of that intermediate.[13]

Koshland Mechanism

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In an early debate in 1969, Dahlquist proposed a covalent mechanism for lysozyme based on kinetic isotope effect[14], but for a long time the ionic mechanism was more accepted. In 2001, a revised mechanism was proposed by Vocadlo via a covalent but not ionic intermediate. Evidence from ESI-MS analysis indicated a covalent intermediate. A 2-Fluorine substituted substrate was used to lower reaction rate and accumulate an intermediate for characterization.[15]The amino acid side-chains glutamic acid 35 (Glu35) and aspartate 52 (Asp52) have been found to be critical to the activity of this enzyme. Glu35 acts as a proton donor to the glycosidic bond, cleaving the C-O bond in the substrate, whereas Asp52 acts as a nucleophile to generate a glycosyl enzyme intermediate. The Glu35 reacts with water to form hydroxyl ion, a stronger nucleophile than water, which then attacks the glycosyl enzyme intermediate, to give the product of hydrolysis and leaving the enzyme unchanged.[16] This covalent mechanism was named after Koshland, who first came up with this type of mechanism[17].

More recently, quantum mechanics/ molecular mechanics (QM/MM) molecular dynamics simulations have been using the crystal of HEWL and predict the existence of a covalent intermediate.[18] Evidence for the ESI-MS and X-ray structures indicate the existence of covalent intermediate, but primarily rely on using a less active mutant or non-native substrate. Thus, QM/MM molecular dynamics provides the unique ability to directly investigate the mechanism of wild-type HEWL and native substrate. The calculations revealed that the covalent intermediate from the Koshland mechanism is ~30 kcal/mol more stable than the ionic intermediate from the Phillips mechanism.[18] These calculation demonstrate that the ionic intermediate is extremely energetically unfavorable and the covalent intermediates observed from experiments using less active mutant or non-native substrates provide useful insight into the mechanism of wild-type HEWL.

Two Possible Mechanisms of Lysozyme

Inhibition

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Imidazole derivatives can form a charge-transfer complex with some residues (in or outside active center) to achieve a competitive inhibition of lysozyme.[19] In Gram-negative bacteria, the lipopolysaccharide acts as a non-competitive inhibitior by highly-favored binding with lysozyme.[20]

Further information: Glycoside hydrolase

Enzyme Conformation Changes

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Lysozyme exhibits two conformations an open active state and a closed inactive state. The catalytic relevance was examined with single walled carbon nanotubes (SWCN) field effect transitors (FETs), where a singular lysozyme was bound to the SWCN FET[21]. Electronically monitoring the lysozyme showed two conformations, an open active site and a closed inactive site. In its active state lysozyme is able to processively hydrolyze its substrate, breaking on average 100 bonds at a rate of 15 per second. In order to bind a new substrate and move from the closed inactive state to the open active state requires two conformation step changes, while inactivation requires one step.

Role in disease and therapy

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Lysozyme is part of the innate immune system. Reduced lysozyme levels have been associated with bronchopulmonary dysplasia in newborns.[22] Children pigs fed with human lysozyme milk can recover from diarrheal disease caused by E. coli faster.[23] The concentration of lysozyme in human milk is 1,600 to 3,000 times greater than the concentration in livestock milk.[24] Human lysozyme is more active than hen egg white lysozyme.[25] A transgenic line of goats (with a founder named "Artemis") were developed to produce milk with human lysozyme to protect children from diarrhea if they can't get the benefits of human breastfeeding.[25][24]

Since lysozyme is a natural form of protection from Gram-positive pathogens like Bacillus and Streptococcus,[26] it plays an important rule in immunology of infants in human milk feeding.[27] Whereas the skin is a protective barrier due to its dryness and acidity, the conjunctiva (membrane covering the eye) is, instead, protected by secreted enzymes, mainly lysozyme and defensin. However, when these protective barriers fail, conjunctivitis results.

In certain cancers (especially myelomonocytic leukemia) 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, conditions that may improve or resolve with treatment of the primary malignancy.

Serum lysozyme is much less specific for diagnosis of sarcoidosis than serum Angiotensin Converting Enzyme; however, since it is more sensitive, it is used as a marker of sarcoidosis disease activity and is suitable for disease monitoring in proven cases [28].

Hyperglycemia, such as in diabetes, causes lysozyme glycation, which reduces its antimicrobial properties, thus increasing the infection susceptibility in diabetic patients.

History

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The antibacterial property of hen egg white, due to the lysozyme it contains, was first observed by Laschtschenko in 1909,[29] although it was not until 1922 that the name 'lysozyme' was coined, by Alexander Fleming (1881–1955), the discoverer of penicillin.[30] Fleming first observed the antibacterial action of lysozyme when he treated bacterial cultures with nasal mucus from a patient suffering from a head cold.[30]

The three-dimensional structure of hen egg white lysozyme was described by David Chilton Phillips (1924–1999) in 1965, when he obtained the first 2-ångström (200 pm) resolution model via X-ray crystallography.[31][32] The structure was publicly presented at a Royal Institution lecture in 1965.[33] Lysozyme was the second protein structure and the first enzyme structure to be solved via X-ray diffraction methods, and the first enzyme to be fully sequenced that contains all twenty common amino acids.[34] As a result of Phillips' elucidation of the structure of lysozyme, it was also the first enzyme to have a detailed, specific mechanism suggested for its method of catalytic action.[35][36][37] This work led Phillips to provide an explanation for how enzymes speed up a chemical reaction in terms of its physical structures. The original mechanism proposed by Phillips was more recently revised.[15]

Chemical synthesis

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The first chemical synthesis of a lysozyme protein was attempted by Prof. George W. Kenner and his group at the University of Liverpool in England.[38] This was finally achieved in 2007 by Steve Kent at the University of Chicago who made synthetic functional lysozyme molecule.[39]

Other applications

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Lysozyme crystals have been used to grow other functional materials for catalysis and biomedical applications.[40][41][42]

See also

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References

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  1. ^ Yoshimura K, Toibana A, Nakahama K (January 1988). "Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces cerevisiae". Biochemical and Biophysical Research Communications. 150 (2): 794–801. doi:10.1016/0006-291X(88)90461-5. PMID 2829884.
  2. ^ Peters CW, Kruse U, Pollwein R, Grzeschik KH, Sippel AE (July 1989). "The human lysozyme gene. Sequence organization and chromosomal localization". European Journal of Biochemistry. 182 (3): 507–16. doi:10.1111/j.1432-1033.1989.tb14857.x. PMID 2546758.
  3. ^ Venkataramani S, Truntzer J, Coleman DR (April 2013). "Thermal stability of high concentration lysozyme across varying pH: A Fourier Transform Infrared study". Journal of Pharmacy & Bioallied Sciences. 5 (2): 148–53. doi:10.4103/0975-7406.111821. PMC 3697194. PMID 23833521.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  4. ^ Chandan RC, Shahani KM, Holly RG (October 1964). "Lysozyme Content of Human Milk". Nature. 204 (4953): 76–7. doi:10.1038/204076a0. PMID 14240122. S2CID 4215401.
  5. ^ "Lysozyme, Product information, Sigma-Aldrich" (PDF).
  6. ^ Skujiņš, J.; PuĶite, A.; McLaren, A. D. (1973). "Adsorption and reactions of chitinase and lysozyme on chitin". Molecular and Cellular Biochemistry. 2 (2): 221–228. doi:10.1007/BF01795475. ISSN 0300-8177. PMID 4359167. S2CID 27906558.
  7. ^ Sharon, N. (18 April 1967). "The Chemical Structure of Lysozyme Substrates and Their Cleavage by the Enzyme". Proceedings of the Royal Society of London B: Biological Sciences. 167 (1009): 402–415. doi:10.1098/rspb.1967.0037. ISSN 0962-8452. PMID 4382803. S2CID 31794497.
  8. ^ Höltje, J. V. (1 January 1996). "Lysozyme substrates". EXS. Experientia Supplementum. 75: 105–110. doi:10.1007/978-3-0348-9225-4_7. ISBN 978-3-0348-9952-9. ISSN 1023-294X. PMID 8765297.
  9. ^ Blake CC, Johnson LN, Mair GA, North AC, Phillips DC, Sarma VR (April 1967). "Crystallographic studies of the activity of hen egg-white lysozyme". Proceedings of the Royal Society of London. Series B, Biological Sciences. 167 (1009): 378–88. doi:10.1098/rspb.1967.0035. PMID 4382801. S2CID 35094695.
  10. ^ Dahlquist FW, Rand-Meir T, Raftery MA (October 1969). "Application of secondary alpha-deuterium kinetic isotope effects to studies of enzyme catalysis. Glycoside hydrolysis by lysozyme and beta-glucosidase". Biochemistry. 8 (10): 4214–21. doi:10.1021/bi00838a045. PMID 5388150.
  11. ^ McKenzie HA, White FH (1991). "Lysozyme and alpha-lactalbumin: structure, function, and interrelationships". Advances in Protein Chemistry. 41: 173–315. doi:10.1016/s0065-3233(08)60198-9. ISBN 9780120342419. PMID 2069076.
  12. ^ Warshel A (November 1978). "Energetics of enzyme catalysis". Proceedings of the National Academy of Sciences of the United States of America. 75 (11): 5250–4. doi:10.1073/pnas.75.11.5250. PMC 392938. PMID 281676.
  13. ^ Weber, J. P.; Fink, A. L. (10 October 1980). "Temperature-dependent change in the rate-limiting step of beta-glucosidase catalysis". The Journal of Biological Chemistry. 255 (19): 9030–9032. doi:10.1016/S0021-9258(19)70521-3. ISSN 0021-9258. PMID 6773958.
  14. ^ Dahlquist, F. W.; Rand-Meir, Tsafrira; Raftery, Michael A. (1 October 1969). "Application of secondary .alpha.-deuterium kinetic isotope effects to studies of enzyme catalysis. Glycoside hydrolysis by lysozyme and .beta.-glucosidase". Biochemistry. 8 (10): 4214–4221. doi:10.1021/bi00838a045. ISSN 0006-2960. PMID 5388150.
  15. ^ a b Vocadlo DJ, Davies GJ, Laine R, Withers SG (August 2001). "Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate" (PDF). Nature. 412 (6849): 835–8. doi:10.1038/35090602. PMID 11518970. S2CID 205020153.
  16. ^ Grisham CM, Garrett RH (2007). "Chapter 14: Mechanism of enzyme action". Biochemistry. Australia: Thomson Brooks/Cole. pp. 467–9. ISBN 978-0-495-11912-8.
  17. ^ Koshland, D. E. (1 November 1953). "Stereochemistry and the Mechanism of Enzymatic Reactions". Biological Reviews. 28 (4): 416–436. doi:10.1111/j.1469-185X.1953.tb01386.x. ISSN 1469-185X. S2CID 86709302.
  18. ^ a b Bowman, Anna L.; Grant, Ian M.; Mulholland, Adrian J. (18 September 2008). "QM/MM simulations predict a covalent intermediate in the hen egg white lysozyme reaction with its natural substrate". Chemical Communications (37): 4425–4427. doi:10.1039/b810099c. ISSN 1364-548X. PMID 18802578.
  19. ^ Swan, Ian D. A. (1972). "The Inhibition of Hen Egg-white Lysozyme by Imidazole and Indole Derivatives". Journal of Molecular Biology. 65 (1): 59–62. doi:10.1016/0022-2836(72)90491-3. PMID 5063023.
  20. ^ Ohno, N.; Morrison, D. C. (15 March 1989). "Lipopolysaccharide interaction with lysozyme. Binding of lipopolysaccharide to lysozyme and inhibition of lysozyme enzymatic activity". The Journal of Biological Chemistry. 264 (8): 4434–4441. doi:10.1016/S0021-9258(18)83761-9. ISSN 0021-9258. PMID 2647736.
  21. ^ Choi, Yongki; Moody, Issa S.; Sims, Patrick C.; Hunt, Steven R.; Corso, Brad L.; Perez, Israel; Weiss, Gregory A.; Collins, Philip G. (20 January 2012). "Single-Molecule Lysozyme Dynamics Monitored by an Electronic Circuit". Science. 335 (6066): 319–324. doi:10.1126/science.1214824. ISSN 0036-8075. PMC 3914775. PMID 22267809.
  22. ^ Revenis ME, Kaliner MA (August 1992). "Lactoferrin and lysozyme deficiency in airway secretions: association with the development of bronchopulmonary dysplasia". The Journal of Pediatrics. 121 (2): 262–70. doi:10.1016/S0022-3476(05)81201-6. PMID 1640295.
  23. ^ Cooper, Caitlin A.; Klobas, Lydia C. Garas; Maga, Elizabeth A.; Murray, James D. (13 March 2013). "Consuming Transgenic Goats' Milk Containing the Antimicrobial Protein Lysozyme Helps Resolve Diarrhea in Young Pigs". PLOS ONE. 8 (3): e58409. doi:10.1371/journal.pone.0058409. ISSN 1932-6203. PMC 3596375. PMID 23516474.
  24. ^ a b Molteni, Megan (30 June 2016). "Spilled Milk". CASE STUDIES: News Features. UNDARK: TRUTH, BEAUTY, SCIENCE. Retrieved 12 January 2017.
  25. ^ a b Cooper CA, Garas Klobas LC, Maga EA, Murray JD (2013). "Consuming transgenic goats' milk containing the antimicrobial protein lysozyme helps resolve diarrhea in young pigs". PLOS ONE. 8 (3): e58409. doi:10.1371/journal.pone.0058409. PMC 3596375. PMID 23516474.
  26. ^ Microbiology: A human perspective. Nester, Anderson, Roberts, Nester. 5th Ed. 2007
  27. ^ Chandra, R. K. (1 September 1978). "Immunological Aspects of Human Milk". Nutrition Reviews. 36 (9): 265–272. doi:10.1111/j.1753-4887.1978.tb07393.x. ISSN 0029-6643. PMID 362248.
  28. ^ Tomita H, Sato S, Matsuda R, Sugiura Y, Kawaguchi H, Niimi T, Yoshida S, Morishita M (1999). "Serum lysozyme levels and clinical features of sarcoidosis". Lung. 177 (3): 161–7. doi:10.1007/pl00007637. PMID 10192763. S2CID 3999327.
  29. ^ Laschtschenko P (1909). "Über die keimtötende und entwicklungshemmende Wirkung Hühnereiweiß" [On the germ-killing and growth-inhibiting effect chicken egg albumin]. Z. Hyg. InfektKrankh. (in German). 64: 419–427. doi:10.1007/BF02216170. S2CID 456259.
  30. ^ a b Fleming A (1 May 1922). "On a remarkable bacteriolytic element found in tissues and secretions". Proceedings of the Royal Society B. 93 (653): 306–317. doi:10.1098/rspb.1922.0023. JSTOR 80959.
  31. ^ Blake CC, Koenig DF, Mair GA, North AC, Phillips DC, Sarma VR (May 1965). "Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Angstrom resolution". Nature. 206 (4986): 757–61. doi:10.1038/206757a0. PMID 5891407. S2CID 4161467.
  32. ^ Johnson LN, Phillips DC (May 1965). "Structure of some crystalline lysozyme-inhibitor complexes determined by X-ray analysis at 6 Angstrom resolution". Nature. 206 (4986): 761–3. doi:10.1038/206761a0. PMID 5840126. S2CID 10234792.
  33. ^ Johnson LN (November 1998). "The early history of lysozyme". Nature Structural Biology. 5 (11): 942–4. doi:10.1038/2917. PMID 9808036. S2CID 2629199.
  34. ^ Canfield RE (August 1963). "The Amino Acid Sequence of Egg White Lysozyme". The Journal of Biological Chemistry. 238 (8): 2698–707. doi:10.1016/S0021-9258(18)67888-3. PMID 14063294.
  35. ^ Vernon CA (April 1967). "The mechanisms of hydrolysis of glycosides and their revelance to enzyme-catalysed reactions". Proceedings of the Royal Society of London. Series B, Biological Sciences. 167 (1009): 389–401. doi:10.1098/rspb.1967.0036. JSTOR 75680. PMID 4382802.
  36. ^ Rupley JA (April 1967). "The binding and cleavage by lysozyme of N-acetylglucosamine oligosaccharides". Proceedings of the Royal Society of London. Series B, Biological Sciences. 167 (1009): 416–28. doi:10.1098/rspb.1967.0038. JSTOR 75682. PMID 4382804.
  37. ^ Sharon N (April 1967). "The chemical structure of lysozyme substrates and their cleavage by the enzyme". Proceedings of the Royal Society of London. Series B, Biological Sciences. 167 (1009): 402–15. doi:10.1098/rspb.1967.0037. JSTOR 75681. PMID 4382803. S2CID 31794497.
  38. ^ Kenner GW (June 1977). "The Bakerian lecture. Towards synthesis of proteins". Proceedings of the Royal Society of London. Series B, Biological Sciences. 197 (1128): 237–53. doi:10.1098/rspb.1977.0068. PMID 19745. S2CID 170906912.
  39. ^ Durek T, Torbeev VY, Kent SB (March 2007). "Convergent chemical synthesis and high-resolution x-ray structure of human lysozyme". Proceedings of the National Academy of Sciences of the United States of America. 104 (12): 4846–51. doi:10.1073/pnas.0610630104. PMC 1829227. PMID 17360367.
  40. ^ Wei H, Wang Z, Zhang J, House S, Gao YG, Yang L, Robinson H, Tan LH, Xing H, Hou C, Robertson IM, Zuo JM, Lu Y (February 2011). "Time-dependent, protein-directed growth of gold nanoparticles within a single crystal of lysozyme". Nature Nanotechnology. 6 (2): 93–7. doi:10.1038/nnano.2010.280. PMID 21278750.
  41. ^ Sanghamitra NJ, Ueno T (May 2013). "Expanding coordination chemistry from protein to protein assembly". Chemical Communications. 49 (39): 4114–26. doi:10.1039/C2CC36935D. PMID 23211931.
  42. ^ Ueno T (July 2013). "Porous protein crystals as reaction vessels". Chemistry. 19 (28): 9096–102. doi:10.1002/chem.201300250. PMID 23813903.
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