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Contents

Lysozyme

Overview

Lysozyme is an enzyme that inhibits the growth of bacteria through lysis of the cell wall. It can be found in salvia, tears, other bodily secretions. Lysozyme is also present in high concentrations in hen egg whites. Lysozymes small size and high stability makes it ideal for protein structure and function research. Furthermore, the enzyme is easy to purify from egg whites and easy to crystallize, unlike most proteins.

Structure

Egg White Lysozyme

Drag the structure with the mouse to rotate

The lysozyme used to analyze structural features was isolated from the eggs of Gallus gallus(chicken). Alternatives names for this lysozyme include 1,4-beta-N-acetylmuramidase C, Allergen Gal d IV, Allergen=Gal d 4. The European Commission number, or EC number, is 3.2.1.17. The sequence consists of 147 amino acids with a molecular weight of 16kD [1]. Lysozyme is of carbon(gray), nitrogen(blue), oxygenpurple) and sulfur(yellow). This shows the ellipsoidal shape, which has the dimension 30 x 30 x 45 Angstroms. The dominate feature, the cleft for substrate binding, is also clear in this figure.

Secondary Structure

Gallus gallus egg white lysozyme has an alpha+beta fold, consisting of eight alpha helices and a three-stranded antiparallel beta sheet. There is also a large amount of random coils and beta turns. Click to visualize the cartoon portrayal of the enzyme with alpha helices and beta sheets highlighted. The alpha helices are in green and the beta sheets in blue. The random coils are gray. Click for the rainbow color ordered cartoon chain from N to C terminals. Depending on where it was isolated from, not all lysozyme molecules will have the same number of alpha helices and beta sheets.

Disulfide and Hydrogen Bonds

Disulfide bonds are formed by the oxidation of two cysteine residues to form a covalent sulphur-sulphur bond [2]. These interactions are not as important for stability, as they are for insuring correct folding patterns. Lysozyme has four disulphide bonds connecting the of the molecule, which are highlighted in yellow. There are also four disulphide bonds in between the , highlighted in red. The residues surrounding the side chain disulphide bonds are highlighted in yellow.

Hydrogen bonds are essential to protein structure, forming an attractive force between the hydrogen attached to an electronegative atom of one molecule and an electronegative atom of a different molecule[3]. The enzyme has many hydrogen bonds connecting the , these are highlighted red. The hydrogen bonds, in yellow, between the are also numerous.


Active Site

The large cleft that transverses the side of lysozyme is the active site. The two amino acids residues that interact with the bound substrate are Asp52 and Glu35. The lysozyme as and representations show Asp52, in green, and Glu35, in purple, branching off in the ball and stick form. The openness of the secondary representation does not allow cleft identification. The cleft, however, can be viewed from the tertiary structures view of the enzyme. This view also has the green Asp52 and purple Glu35 visible. The peptidoglycan that binds in the lysis reaction is considered a ligand of lysozyme. Lysozyme has no natural ligands.

Polar vs Nonpolar Residues

The hydrophobic collapse is the driving force behind protein folding. The nonpolar residues minimize there contact with water by compacting into a hydrophobic core. For this reason the hydrophobic effect is the major determinant of protein structure and has the greatest influence of stability. Understanding the polar and nonpolar areas of a molecule gives an understanding of where water can and will interact. This visualizes the polar(red) and nonpolar(white) regions of the secondary structure. Using the same labeling, the polar and nonpolar residues are represented in this ball and stick . Through these depictions of lysozyme it is apparent the hydrophobic, nonpolar residues favor the inside of the molecule and the hydrophilic, polar residues tend to stay on the outside. The active site residues are highlighted in this polar vs nonpolar dot . Viewing this depiction makes it clear that Asp52, in yellow, is in a highly polar, hydrophilic area. While Glu35, in green, is located in more of a nonpolar, hydrophobic region. In the lysis reaction, this placement of Glu35 in a hydrophobic area makes it possible for protonation at a neutral pH, because the dissociation is suppressed.

The charged residues are also located on the outside of the enzyme. This representation shows the positive, acidic residues in red and the negative, basic residues in blue. Research has shown that charged residues are major determinants of the transmembrane orientation.

Function

Lysozyme’s main function is to protect from infection. The enzyme is a general non-specific organism defense effective against gram positive bacteria. Lysozyme degrades the polysaccharides found in cell walls by catalyzing the 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[4]. X-ray crystallography has shown that the binding of lysozyme and the substrates slightly deforms both structures. The binding first distorts the fourth hexose in the chain to the half chair conformation [5]. This imposes a strain on the C-O bond on the ring-4 side of the oxygen bridge between rings 4 and 5[6]. The polysaccharide is broken at this point and a molecule of water is inserted between the two hexoses. The reaction mechanism is shown below.

Image:Lys.gif

History

Laschtschenko first described lysozyme in chicken eggs in 1909. In 1919, Bloomfield reported the enzyme in saliva. Not until its discovery by Alexander Fleming in 1922 was lysozyme officially named and understood[7]. Researching medical antibiotics, Fleming tested human mucus on a live culture. To his surprise, it successfully killed the bacteria. The phenomena was carefully analyzed and, shortly after, proven that lysozyme was the main active enzyme. Fleming had discovered one of the human body’s natural defenses against infection. Lysozyme could not successfully be used as an antibiotic, however, because its large size inhibits transportation through cells[8]. The enzyme has been put to good use, being the source of much protein structure and function research.

As mentioned earlier lysozyme can be purified from hen egg-whites and crystallized quite simply. This has made it the best object for X-Ray analysis for many years. The X-Ray beam diffraction of lysozyme crystals has an extremely high resolution, reaching 0.94 Angstroms. In 1965 David Chilton Phillips successfully solved the structure through X-Ray analysis with 2 angstrom resolution[9]. Lysozyme was the first enzyme ever to have its structure solved. A year later, the mechanism was explained. Today lysozyme is still being used in research and is commercially valuable enzyme used for many purposes, including the treatment of ulcers and infections, and as a food and drug preservative.


References

  1. ↑ Lysozyme cprecursor - gallus gallus (chicken). (2010, October 5). Retrieved from http://www.uniprot.org/uniprot/P00698
  2. ↑ Day, A. (1996). Disulphide bonds. Retrieved from http://www.cryst.bbk.ac.uk/PPS2/projects/day/TDayDiss/DisulphideBonds.html
  3. ↑ Ophardt, C. (2003). Intermolecular forces: hydrogen bonds. Retrieved from http://www.elmhurst.edu/~chm/vchembook/161Ahydrogenbond.html
  4. ↑ Lysozyme. (2008). Retrieved from http://lysozyme.co.uk/
  5. ↑ Voet, D, G., J, & W., C. (2008). Fundamentals of biochemistry: life at the molecular level. John Wiley & Sons Inc
  6. ↑ Kimball, J. (2010, May 26). Enzymes. Retrieved from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html#lysozyme
  7. ↑ Worthington, K. (2010). Lysozyme. Retrieved from http://www.worthington-biochem.com/ly/default.html
  8. ↑ Goodsell, D. (2000, September). Lysozyme. Retrieved from http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb9_1.html
  9. ↑ Lysozyme. (2008). Retrieved from http://lysozyme.co.uk/
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