Lysozyme

STRUCTURE AND MECHANISM:

Enzyme Classification Number: EC 3.2.1.17

The Russian scientist P. Laschtchenko first described the bacteriolytic properties of hen egg white lysozyme in 1909. In 1922, Alexander Fleming, the London bacteriologist who later discovered penicillin, gave the name lysozyme to the agent in mucus and tears that destroyed certain bacteria. Lysozyme was the first enzyme for which the X-ray structure was determined at high resolution. This was achieved in 1965 by David Phillips, working at the Royal Institution in London. Phillips went on to propose a mechanism for lysozyme action that was based principally on structural data. The Phillips mechanism has since been borne out by experimental evidence, as we shall see later.

Lysozyme is found widely in the cells and secretions (including tears and saliva) of vertebrates, and hen egg white is particularly rich in this enzyme. .Lysozyme catalyses the hydrolysis of glycosidic bonds that link C-1 of N-acetylmuramic acid (NAM) and C-4 of N-acetylglucosamine (NAG) in polysaccharides of bacterial cell walls. In doing so, it damages the integrity of the cell wall and thereby acts as a bacteriocidal agent. The NAM–NAG bond is represented in Figure 1, with the site of cleavage by lysozyme indicated.

 Figure 1: Part of the polysaccharide component of bacterial cell walls, showing the alternating N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) residues. This polysaccharide is a substrate for lysozyme, which hydrolyses the glycosidic bond at the position indicated. (For clarity, and to permit a linear representation of the molecule, some of the bonds are shown in a zig-zag form.)

Lysozyme is a relatively small enzyme. It is a globular-ellipsoidal protein with dimensions of 30 x 30 x 45 A°. Hen egg white lysozyme consists of a single polypeptide of 129 amino acids in length (Figure 2) with M_r 14 600. From X-ray diffraction data, we can see that there is a distinct cleft in the lysozyme structure ( Figure  3). It has eight cysteine residues linked in four disulfide bonds.The residues important for the catalysis are aspartate-52 and glutamate-35 in the cleft of the enzyme where the substrate is predicted to bind The active site is located in this cleft. In the amino acid sequence in  Figure  2 and in the space-filling model of lysozyme in  Figure  3, those residues that line the substrate binding pocket in the folded protein have been highlighted.

Figure 2: The amino acid sequence of hen egg white lysozyme, with the residues that line the substrate binding pocket highlighted in grey. Asp 52 and Glu 35, key residues in the active site, are highlighted in red and yellow respectively.

Figure 3: A space-filling model of hen egg white lysozyme in which key residues have been highlighted. Asp 52 is in red; Glu 35 is in yellow; some of the residues lining the substrate binding pocket are shown in grey.

The active site of lysozyme is a long groove that can accommodate six sugars of the polysaccharide chain at a time. On binding the polysaccharide, the enzyme hydrolyses one of the glycosidic bonds. If the six sugars in the stretch of polysaccharide are identified as A–F, the cleavage site is between D and E, as indicated in  Figure 1. The two polysaccharide fragments are then released.  Figure  4 depicts the stages of this reaction, which are also described in detail below.

Figure 4: The catalytic mechanism of lysozyme. Note that only key residues involved in catalysis (Glu 35 and Asp 52) are shown. The stages are described in detail in the text. (Based on Phillips, 1966)

Long description

1.      On binding to the enzyme, the substrate adopts a strained conformation. Residue D is distorted (not shown in the diagram) to accommodate a –CH₂OH group that otherwise would make unfavourable contact with the enzyme. In this way, the enzyme forces the substrate to adopt a conformation approximating to that of the transition state.

2.      Residue 35 of the enzyme is glutamic acid (Glu 35) with a proton that it readily transfers to the polar O atom of the glycosidic bond. In this way, the C–O bond in the substrate is cleaved ( Figure  4a and b).

3.      Residue D of the polysaccharide now has a net positive charge; this reaction intermediate is known as an oxonium ion ( Figure  4b). The enzyme stabilises this intermediate in two ways. Firstly, a nearby aspartate residue (Asp 52), which is in the negatively charged carboxylate form, interacts with the positive charge of the oxonium ion. Secondly, the distortion of residue D enables the positive charge to be shared between its C and O atom. (Note that this sharing of charge between atoms is termed resonance in the same way as the sharing of electrons between the atoms of the peptide group.) Thus the oxonium ion intermediate is the transition state. Normally, such an intermediate would be very unstable and reactive. Asp 52 helps to stabilise the oxonium ion, but it does not react with it. This is because, at 3 Å distance, the reactive groups are too far apart.

4.      The enzyme now releases residue E with its attached polysaccharide, yielding a glycosyl-enzyme intermediate. The oxonium ion reacts with a water molecule from the solvent environment, extracting a hydroxyl group and re-protonating Glu 35 ( Figure  4c and Figure 4d).

5.      The enzyme then releases residue D with its attached polysaccharide and the reaction is complete.

·         The catalytic mechanism of lysozyme involves both general acid and general base catalysis. Which residues participate in these events?

·         Glu 35 participates in general acid catalysis (donates a proton) and Asp 52 participates in general base catalysis (stabilising the positive charge of the oxonium ion).

The Phillips mechanism for lysozyme catalysis, as outlined above, is supported by a number of experimental observations. In particular, the importance of Glu 35 and Asp 52 in the process has been confirmed by site-directed mutagenesis (SDM) experiments. SDM is a very powerful technique for examining the role of individual amino acid residues in a protein's function. SDM involves the use of recombinant DNA technology to selectively replace the residue of interest with a different amino acid with critically different properties. The resulting protein can then be tested functionally, e.g. with respect to substrate binding or catalytic activity. When this technique was applied to lysozyme to replace Glu 35 with a glutamine residue (Gln), the resulting protein could still bind the substrate (albeit less strongly) but it had no catalytic activity. Glu 35 is therefore essential for lysozyme's catalytic activity. When Asp 52 was replaced with an asparagine (Asn) residue, the mutant protein had less than 5% of the catalytic activity of normal (wild-type) lysozyme, in spite of the fact that the mutant form actually had a twofold higher affinity for the substrate. It follows that Asp 52 is essential for lysozyme's catalytic activity. Experiments using chemical agents that covalently modified these residues, without significantly affecting the X-ray structure, similarly proved that they were essential for catalytic activity.