Human  Lysozyme

 

Introduction 

     Human lysozyme was discovered more than seventy years ago in 1922 by Alexander Fleming.  Alexander Fleming was a bacteriologist in London and one day either accidentally or deliberately he allowed a few drops of nasal mucous to drop on a petri dish colonated with bacteria.  After several hours he looked at the dish and observed clear areas where the bacteria had been destroyed.  He named the substance in the mucous lysozyme. (lyso-because it lysed bacteria and zyme because it proved to be an enzyme)[Stryer,1981]
     Lysozyme is widely distributed in biological systems; it is found in both plants and animals.  In humans, it is found in abundance in tears, saliva leukocytes, and serum.[Oserman, 1969]  Fleming was disappointed to find that lysozyme wasn't effective against very harmful bacteria but he did discover one substance that proved to be a powerful antibiotic - penicillin.
     Lysozyme does not actually lyse a bacteria out right, but rather it attacks the polysaccharide coat which makes up the cell wall.  By breaking down and weakening the cell wall, the cell will be unable to resist osmotic pressure.  The build up of pressure will then cause the cell to lyse.  Unlike many proteins lysozyme does not contain any prosthetic groups for catalysis like heme in hemoglobin.  Human lysozyme itself is a relatively simple structure made up of 130 residues.[Hooke, et al., 1994]  The number of residues for lysozyme varies in different biological systems. For example the popular hen egg white lysozyme is only 129 residues.
    To see a space fill model of the enzyme press here.  
    To see a cartoon model of the enzyme press here. 

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Reaction Mechanism

     Before getting into the reaction mechanism of lysozyme it important to review the structure of the ligand that lysozyme cleaves.  Like pac man eating little white dots, lysozyme likes to much (catalyze) the reaction that breaks a specific polysaccharide apart.  A polysaccharide is nothing more than the linking together of sugar residues to form a chain.  The polysaccharide of lysozyme reaction is made up of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).  NAM and NAG are joined together by glycosidic linkage.  This linkage comes in two forms.  Either as an alpha configuration where the linking oxygen comes from an OH group below the plane of the sugar ring (Fig 1) or a beta configuration where the oxygen comes from an OH group above the plane of the sugar ring (Fig 2)  In figures 1 and 2, the linking oxygen between NAM and NAG comes from an OH- group on NAG. The type of bond that lysozyme cleaves is called a beta (1-4) NAM-NAG glycosidic bond.  More accurately it is the glycosidic bond between the C-1 of NAM and the oxygen attached to the C-4 of NAG. [Stryer, 1981]  The portion of the polysaccharide ligand that lysozyme binds to contains six alternating NAM and NAG rings labeled A through F.
    The polysaccharide chains in the cell wall of bacteria are also linked together side by side to form an interlinked sheet.  These side by side chains are held together via bonds between the N-acetylmuramic acid residues.  Another ligand that is cleaved by lysozyme is chitin, which is found in the shells of crustaceans.  Chitin does not have any NAM sugars but rather is made entirely of NAG sugars.
     The reaction mechanism of human lysozyme occurs at its peak at a pH of 5.1.  If the pH is lowered to 4.0, the rate drops 60%.[Song, 1994]  The actual reaction mechanism that takes place is that a proton is transferred from the carboxyl group of Glu 35 to the bond between C-1 of the D ring and the glycosidic oxygen atom.(Fig. 3)  This proton transfer results a carbonium ion forming on C-1 of the D ring.  Aspartic acid 52 helps to stabilize the carbonium ion formed.  Press to see where Glu 35 and Asp 52 are on the enzyme.   Now that the bond between NAM and NAG is broken the E and F rings of the ligand diffuse away.  The carboxyl group on Glu 35 then regains a proton from the solvent while the carbonium ion of the D ring reacts with OH- of the solvent to remove the ionic charge. (Fig. 4)  The remaining four ring members then diffuses away from the active site freeing it for another reaction to take place.


Figure 1 Alpha glycosidic bond between NAM & 
               NAG. [Stryer, 1981]


Figure 2 Beta glycosidic bond between NAM &
              NAG. [Stryer, 1981]


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-Green is for Aspartic acid 53
-Yellow is for Glutamic acid 35

 

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Figure 3  Reaction mechanism of glycosidic bond cleavage by lysozyme. [Stryer, 1981]

 


Figure 4  Removal of the carbonium ion on NAG and reprotonating Glu 35. [Stryer, 1981]

Structure: Overview

     Human lysozyme (HL) is made up of 130 amino acids and has a macromolecular weight of 14,691daltons, calculated by electrospray ionization mass spectrometry.[Booth et al., 1997]  It is a single strand enzyme with no subunits.  The secondary structures of HL can be broken down into seven helices and one beta sheet consisting of three strands.
     The seven helices are made up of residues 5-14, 25-35,81-85,90-100,105-108, 110-115, 122-124.[PDB 1LZ1]  Press here to view the seven helices.   Note two of the seven helices are colored blue. Press here to view the helices in the overall model.   The three strands of the beta sheet are made up of residues 43-46, 51-54, and 59-60.  Press here to view the beta sheet.   Press here to see the beta sheet in the overall model  
     There are four intramolecular disulfide bonds between cysines which help to maintain the folded structure of HL.  These bonds are between cystine 6 and 128,  cystine 30 and 116,  cystine 65 and 81,  and cystine 77 and 95.  ( The first cystine of each pair is colored green and the second is colored yellow.)  To see all four disulfide bonds on one molecule press here.   Below is figure 5 which shows the residue sequence of HL and allows for visualization of where the secondary structures are.