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Lysozyme - Hen Egg White (HEW)

Lysozyme is an enzyme that breaks down cell walls by hydrolyzing certain glycosidic linkages in the peptidoglycan of cell walls. It is found in the cells and secretions of various vertebrates, and likely functions as an bacteria-killing or -disposal agent. The lysozyme found in hen egg white has been investigated more thoroughly than other species of lysozyme, and so is better understood then most enzymes.[1] The PDB code of HEW lysozyme shown on this page is 3IJU and the assigned EC number for lysozyme is EC 3.2.1.17.

History

The enzyme Lysozoyme was first discovered and named in 1922 by Alexander Fleming. Fleming contaminated petri plates containing gram-positive bacteria with nasal mucous. After this contamination, he found that the bacterial cells on the petri plates had been lysed. He continued to investigate the cause of the cell lysis, and discovered lysozyme.[2] He also discovered that lysozyme only works on gram-positive cells, and not on gram-negative cells. Gram-negative cells have an outer cell membrane containing lipopolysaccharides, which cannot be digested by lysozyme.[3] Gram-positive cells, however, do not have this outer membrane, and so can be lysed by lysozyme.

The structure of HEW Lysozyme was investigated and found out in 1965 by David Phillips, making it the first enzyme to have its structure determined. Phillips initially elucidated the structure through X-Ray crystallography and then continued his investigation of substrate binding by building models of the enzyme.It was through this larger-scale model building that Phillips was able to identify the catalytic site of lysozyme.[4]

Structure

Basic Structure of HEW Lysozyme.

Drag the structure with the mouse to rotate

Basics

HEW Lysozyme is a small enzyme, weighing 14.3 kD and containing only 129 amino acid residues. From his determination of the structure, Phillips found that lysozyme had an ellipsoidal shape and a prominent cleft which acted as the sustrate-enzyme binding site. This can be seen best when looking at a surface view of the protein. The two amino acids that most directly interact with the substrate when cutting glycosidic bonds are (Glu is blue and Asp is purple).

Secondary Structures

The of HEW lysozyme are split into two groups. First, there are seven alpha helices (in green), which are in random coils. Second there is one beta sheet (in blue), which contains three antiparallel strands.

Intermolecular Forces

In general, there are many intermolecular forces within the structure of lysozyme, two of these being disulfide bonds and hydrogen bonds. HEW lysozyme has eight total disulfide bonds- four are , and the other four are . HEW lysozyme has numerous hydrogen bonds between its , and even more between its . These bonds help to stabilize the enzyme and hold it together in the correct orientation.

Catalytic Reaction

Lysozyme catalyzes a hydrolysis reaction between polysaccharides. In particular, it hydrolyzes the beta (1 to 4) linkage between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). The reaction involves Gln 57 acting as an acid catalyst and is characterized by a covalent intermediate containing Asp 52.

Mechanism

To begin, the substrate starts to hydrogen bind to sites A-F, but the NAM cannot bind at site D in its normal chair conformation because of its interaction with other side chains. The steric hindrance caused by this predicament is solved by the NAM flipping into a half-chair conformation, making it able to hydrogen bind correctly. Then Glu 35 acts as an acid catalyst, giving up a proton to break the glycosidic bond and forming an oxonium ion. Asp 52 attacks the oxonium ion and forms a covalent intermediate.Then water comes in and binds to the covalent intermediate, detaching it from the Asp 52. The substrate has undergone base catalysis and separates from the enzyme binding site.[5] An image of the mechanism can be found here: ,[[1]]

Inhibitors

Lysozyme is best inhibited by small saccharides which act competitively with the natural substrate. The smaller saccharides will bind to the first three binding sites of the cleft (sites A-C), but not reach sites D and E, where the enzyme cuts the glycosidic bond. So, the competitive inhibitor will stick in the cleft, not allowing the substrate to bind to the enzyme complex.[6] Several known inhibitors of lysozyme are SDS, N-acetyl-D-glucosamine, and various alcohols and oxidizing agents.[7]

Current Uses

Lysozyme is frequently used in hydrolysis of bacterial cell walls and of chitin, protein purification, and nucleic acid and plasmid preparation. Recent areas of research include further investigation of gene regulation of lysozyme, better elucidation of its secondary structure, and "refining its use in biochemical applications".[8]

References

  1. ↑ Voet, D., Voet, J., Pratt, C.(2008) Fundamentals of Biochemistry: Life at the Molecular Level, 3rd edition. John Wiley and Sons, Inc.
  2. ↑ copyright 2006-2008. http://lysozyme.co.uk/
  3. ↑ http://en.wikipedia.org/wiki/Gram-negative_bacteria
  4. ↑ Voet, D., Voet, J., Pratt, C.(2008) Fundamentals of Biochemistry: Life at the Molecular Level, 3rd edition. John Wiley and Sons, Inc.
  5. ↑ Voet, D., Voet, J., Pratt, C.(2008) Fundamentals of Biochemistry: Life at the Molecular Level, 3rd edition. John Wiley and Sons, Inc.
  6. ↑ http://mcdb-webarchive.mcdb.ucsb.edu/sears/biochemistry/tw-enz/lysozyme/HEWL/lysozyme-overview.htm
  7. ↑ http://www.worthington-biochem.com/ly/default.html
  8. ↑ http://www.worthington-biochem.com/ly/default.html
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