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LYSOZYME STRUCTURE TUTORIAL

In 1922 Alexander Fleming while working in his laboratory even though he had a cold, accidentally added a few drops of nasal mucus to a bacterial culture - So much for systematic research. To his surprise something in the mucus killed the bacteria. The active substance was the enzyme lysozyme.

Lysozyme serves as a model of protein structure and function. Lysozyme is a component of tears and mucous and is believed to serve a protective role in defending the body against productive bacterial infection. Lysozyme disrupts bacteria by hydrolyzing the mucopolysaccharide portion of the cell wall. Specifically, the enzyme hydrolyzes the 1-4 glycosidic linkage between alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues. The enzymatic mechanism of the hydrolysis is well described.

NAG	NAM

In this tutorial we will examine several features of the enzyme, including the cleft in which the NAG binds. In CH 313 laboratory, we obtained lysozyme from hen egg white (HEW), which is an excellent source of the enzyme due to its overwhelming abundance. Lysozyme from turkey or quail eggs has also been crystallized and investigated thoroughly.

Lysozyme was the second protein and the first enzyme to have its detailed molecular structure worked out by x-ray analysis. HEW lysozyme is a single polypeptide of 129 amino acids. It has a mass of 14.6 kDa and contains four disulfide bonds. The first X-ray structure of HEW lysozyme, made by David Phillips in 1965, revealed its ellipsoidal shape as well as the prominent cleft that forms across the face of the enzyme. The cleft forms the substrate binding site and positions the two catalytic functional groups, Glu35 and Asp 52, on opposing sides of the NAM-NAG bond.

Note: The following tutorial was made to be completed along with the molecular viewing program, RasMol. Although not necessary, it may be helpful to learn more about RasMol's Options.

Before we examine the lysozyme molecule, if you would like to view the divers structures through rasmol, it is first necessary to view the entire lysozyme molecule with the NAG.

The first image to analyze is lysozyme molecule without its NAG substrate. With the already loaded image, type in the command window:

select amino
color group
cartoons on
wireframe off
select 1
color purple
wireframe 80
select 129
color red
select nag
wireframe off
select amino
rotate x -80
rotate y -40
rotate z -45

This image is displayed in cartoon ribbons, with a group color scheme. Group colors allow the molecule to be displayed from the C terminus to the N terminus following the rainbow from red to violet. Notice the C terminus in red and N terminus in violet.

Next we will examine the several alpha helices as well as the three stranded anti-parallel beta sheets. Type:

select alpha
color cyan
select helices
color red
select sheets
color green

Notice the three regular helices in red and the three main beta sheets in green. Notice the two closer antiparallel pleated sheets. They account for many hydrogen bonds not found in such quantity in the more irregular third sheet. Also if we look at the molecule with disulfide bridges we can correlate the four bridges with the architecture of the helices and sheets.Type:

select cys
color [255,255,0]
wireframe 150
ssbond 150

Furthermore, if we examine the location of the cleft, in yellow, overlaying the helices and sheets, we notice the cleft actually traverses all three beta sheets.

select amino
wireframe on
select 34-37, 44, 52, 56, 57, 59, 62, 63, 75, 101, 103, 107-110, 114
color yellow

Now let us examine the cleft alone in yellow. The two red functional groups, Glu 35 and Asp 52, again, are the reactive center's catalytic groups. Type:

select amino
ribbons off
ssbonds off
wireframe on
select 34-37, 44, 52, 56, 57, 59, 62, 63, 75, 101, 103, 107-110, 114
spacefill
color yellow
select hetero
spacefill off
select 35, 52
color red
When you are ready to continue, type: quit to continue.

Before we continue to examine the other properties of lysozyme, we must look at the NAG substrate. The hen egg white's tri N-acetylglucosamine is a simple structure that fits into the right side of the cleft. Here is the substrate alone: nag.ent. Notice the three glucosamines, a prominent component of bacteria cell walls.

When you are ready, type: quit to continue.

Finally, we see the lysozyme molecule with the NAG, in red, in the cleft:pdb1hew.ent Notice how it fits into only a small corner of the cleft (in yellow). Larger oligosaccharide substates fill a greater part of the cleft. Type:

select 34-37, 44, 52, 56, 57, 59, 62, 63, 75, 101, 103, 107-110, 114
color yellow
spacefill
select hetero
spacefill off
select nag
color red
spacefill
select 35, 52
color green
rotate x -80
rotate y -40
rotate z -45

Now we will examine the acidic properties of lysozyme. The acidic functional groups are in yellow, and note the two acidic catalytic residues here in purple. Obviously, this molecule is only slightly acidic. Of the 120 amino acid residues, there are only 8 Aspartic acids and only 2 Glutamic acids. Type:

select all
color chain
spacefill
select hetero
spacefill off
select acidic
color yellow
select 35, 52
color purple

If we look at the basic properties of the molecule, again in yellow, not only do we notice that there are more basic residues included, but by typing slab 50, slab 40, slab 70, and finally slab off, we see the basic residues stick predominantly to the outide of the structure. Again, the NAG is in red here. Type:

select amino
color group
select basic
color yellow

Next we focus on the hydrophobicity of the protein. Note the purple hydrophobic residues. Lysozyme agrees with the standard "hydrophobic in, hydrophillic out" protein model. Type slab 50, slab 70, and slab off to find out where the majority of the hydrophobic residues reside. Type:

select amino
color group
select hydrophobic
color purple
Finally, type:quit