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 N-acetylmuramic acid (NAM) and 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 40, with the site of cleavage by lysozyme indicated.
Lysozyme is a relatively small enzyme. Hen egg white lysozyme consists of a single polypeptide of 129 amino acids in length (Figure 41) with Mr 14 600. From X-ray diffraction data, we can see that there is a distinct cleft in the lysozyme structure (Figure 42). The active site is located in this cleft. In the amino acid sequence in Figure 41 and in the space-filling model of lysozyme in Figure 42, those residues that line the substrate binding pocket in the folded protein have been highlighted.
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 40. The two polysaccharide fragments are then released. Figure 43 depicts the stages of this reaction, which are also described in detail below.
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.
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 43a and b).
Residue D of the polysaccharide now has a net positive charge; this reaction intermediate is known as an oxonium ion (Figure 43b). 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.
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 43c and d).
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 and will be discussed in some detail in Section 7.2. 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.