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.

Classification and Nomenclature of Enzymes by the Reactions they Catalyze

Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)

Enzyme Nomenclature

1. General principles

Because of their close interdependence, it is convenient to deal with the classification and nomenclature together.

The first general principle of these 'Recommendations' is that names purporting to be names of enzymes, especially those ending in -ase, should be used only for single enzymes, i.e. single catalytic entities. They should not be applied to systems containing more than one enzyme. When it is desired to name such a system on the basis of the overall reaction catalyzed by it, the word system should be included in the name. For example, the system catalyzing the oxidation of succinate by molecular oxygen, consisting of succinate dehydrogenase, cytochrome oxidase, and several intermediate carriers, should not be named succinate oxidase, but it may be called the succinate oxidase system. Other examples of systems consisting of several structurally and functionally linked enzymes (and cofactors) are the pyruvate dehydrogenase system, the similar 2-oxoglutarate dehydrogenase system, and the fatty acid synthase system.

In this context it is appropriate to express disapproval of a loose and misleading practice that is found in the biological literature. It consists in designation of a natural substance (or even of an hypothetical active principle), responsible for a physiological or biophysical phenomenon that cannot be described in terms of a definite chemical reaction, by the name of the phenomenon in conjugation with the suffix -ase, which implies an individual enzyme. Some examples of such phenomenase nomenclature, which should be discouraged even if there are reasons to suppose that the particular agent may have enzymic properties, are: permease, translocase, reparase, joinase, replicase, codase, etc..

The second general principle is that enzymes are principally classified and named according to the reaction they catalyze. The chemical reaction catalyzed is the specific property that distinguishes one enzyme from another, and it is logical to use it as the basis for the classification and naming of enzymes.

Several alternative bases for classification and naming had been considered, e.g. chemical nature of the enzymes (whether it is a flavoprotein, a hemoprotein, a pyridoxal-phosphate protein, a copper protein, and so on), or chemical nature of the substrate (nucleotides, carbohydrates, proteins, etc.). The first cannot serve as a general basis, for only a minority of enzymes has such identifiable prosthetic groups. The chemical nature of the enzyme has, however, been used exceptionally in certain cases where classification based on specificity is difficult, for example, with the peptidases (subclass EC 3.4). The second basis for classification is hardly practicable, owing to the great variety of substances acted upon and because it is not sufficiently informative unless the type of reaction is also given. It is the overall reaction, as expressed by the formal equation that should be taken as the basis. Thus, the intimate mechanism of the reaction, and the formation of intermediate complexes of the reactants with the enzyme is not taken into account, but only the observed chemical change produced by the complete enzyme reaction. For example, in those cases in which the enzyme contains a prosthetic group that serves to catalyze transfer from a donor to an acceptor (e.g. flavin, biotin, or pyridoxal-phosphate enzymes) the name of the prosthetic group is not normally included in the name of the enzyme. Nevertheless, where alternative names are possible, the mechanism may be taken into account in choosing between them.

A consequence of the adoption of the chemical reaction as the basis for naming enzymes is that a systematic name cannot be given to an enzyme until it is known what chemical reaction it catalyzes. This applies, for example, to a few enzymes that have so far not been shown to catalyze any chemical reaction, but only isotopic exchanges; the isotopic exchange gives some idea of one step in the overall chemical reaction, but the reaction as a whole remains unknown.

A second consequence of this concept is that a certain name designates not a single enzyme protein but a group of proteins with the same catalytic property. Enzymes from different sources (various bacterial, plant or animal species) are classified as one entry. The same applies to isoenzymes (see below). However, there are exceptions to this general rule. Some are justified because the mechanism of the reaction or the substrate specificity is so different as to warrant different entries in the enzyme list. This applies, for example, to the two cholinesterases, EC 3.1.1.7 and 3.1.1.8, the two citrate hydro-lyases, EC 4.2.1.3 and 4.2.1.4, and the two amine oxidases, EC 1.4.3.4 and 1.4.3.6. Others are mainly historical, e.g. acid and alkaline phosphatases (EC 3.1.3.1 and EC 3.1.3.2).

A third general principle adopted is that the enzymes are divided into groups on the basis of the type of reaction catalyzed, and this, together with the name(s) of the substrate(s) provides a basis for naming individual enzymes. It is also the basis for classification and code numbers.

Special problems attend the classification and naming of enzymes catalyzing complicated transformations that can be resolved into several sequential or coupled intermediary reactions of different types, all catalyzed by a single enzyme (not an enzyme system). Some of the steps may be spontaneous non-catalytic reactions, while one or more intermediate steps depend on catalysis by the enzyme. Wherever the nature and sequence of intermediary reactions is known or can be presumed with confidence, classification and naming of the enzyme should be based on the first enzyme-catalyzed step that is essential to the subsequent transformations, which can be indicated by a supplementary term in parentheses, e.g. acetyl-CoA:glyoxylate C-acetyltransferase (thioester-hydrolysing, carboxymethyl-forming) (EC 2.3.3.9, cf. section 3).

To classify an enzyme according to the type of reaction catalyzed, it is occasionally necessary to choose between alternative ways of regarding a given reaction. Some considerations of this type are outlined in section 3 of this chapter. In general, that alternative should be selected which fits in best with the general system of classification and reduces the number of exceptions.

One important extension of this principle is the question of the direction in which the reaction is written for the purposes of classification. To simplify the classification, the direction chosen should be the same for all enzymes in a given class, even if this direction has not been demonstrated for all. Thus the systematic names, on which the classification and code numbers are based, may be derived from a written reaction, even though only the reverse of this has been actually demonstrated experimentally. In the list in this volume, the reaction is written to illustrate the classification, i.e. in the direction described by the systematic name. However, the common name may be based on either direction of reaction, and is often based on the presumed physiological direction.

Many examples of this usage are found in section 1 of the list. The reaction for EC 1.1.1.9 is written as an oxidation of xylitol by NAD⁺, in parallel with all other oxidoreductases in subgroup EC 1.1.1, and the systematic name is accordingly, xylitol:NAD⁺ 2-oxidoreductase (D-xylulose-forming). However, the common name, based on the reverse direction of reaction, is D-xylulose reductase.

2. Common and Systematic Names

The first Enzyme Commission gave much thought to the question of a systematic and logical nomenclature for enzymes, and finally recommended that there should be two nomenclatures for enzymes, one systematic, and one working or trivial. The systematic name of an enzyme, formed in accordance with definite rules, showed the action of an enzyme as exactly as possible, thus identifying the enzyme precisely. The trivial name was sufficiently short for general use, but not necessarily very systematic; in a great many cases it was a name already in current use. The introduction of (often cumbersome) systematic names was strongly criticized. In many cases the reaction catalyzed is not much longer than the systematic name and can serve just as well for identification, especially in conjunction with the code number.

The Commission for Revision of Enzyme Nomenclature discussed this problem at length, and a change in emphasis was made. It was decided to give the trivial names more prominence in the Enzyme List; they now follow immediately after the code number, and are described as Common Name. Also, in the index the common names are indicated by an asterisk. Nevertheless, it was decided to retain the systematic names as the basis for classification for the following reasons:

(i) The code number alone is only useful for identification of an enzyme when a copy of the Enzyme List is at hand, whereas the systematic name is self-explanatory;

(ii) The systematic name stresses the type of reaction; the reaction equation does not;

(iii) Systematic names can be formed for new enzymes by the discoverer, by application of the rules, but code numbers should not be assigned by individuals;

(iv) Common names for new enzymes are frequently formed as a condensed version of the systematic name; therefore, the systematic names are helpful in finding common names that are in accordance with the general pattern.

It is recommended that for enzymes that are not the main subject of a paper or abstract, the common names should be used, but they should be identified at their first mention by their code numbers and source. Where an enzyme is the main subject of a paper or abstract, its code number, systematic name, or, alternatively, the reaction equation and source should be given at its first mention; thereafter the common name should be used. In the light of the fact that enzyme names and code numbers refer to reactions catalyzed rather than to discrete proteins, it is of special importance to give also the source of the enzyme for full identification; in cases where multiple forms are known to exist, knowledge of this should be included where available.

When a paper deals with an enzyme that is not yet in the Enzyme List, the author may introduce a new name and, if desired, a new systematic name, both formed according to the recommended rules. A number should be assigned only by the Nomenclature Committee of IUBMB.

The Enzyme List contains one or more references for each enzyme. It should be stressed that no attempt has been made to provide a complete bibliography, or to refer to the first description of an enzyme. The references are intended to provide sufficient evidence for the existence of an enzyme catalyzing the reaction as set out. Where there is a major paper describing the purification and specificity of an enzyme, or a major review article, this has been quoted to the exclusion of earlier and later papers. In some cases separate references are given for animal, plant and bacterial enzymes.

3. Scheme for the classification of enzymes and the generation of EC numbers

The first Enzyme Commission, in its report in 1961, devised a system for classification of enzymes that also serves as a basis for assigning code numbers to them. These code numbers, prefixed by EC, which are now widely in use, contain four elements separated by points, with the following meaning:

(i) The first number shows to which of the six main divisions (classes) the enzyme belongs,

(ii) The second figure indicates the subclass,

(iii) The third figure gives the sub-subclass,

(iv) The fourth figure is the serial number of the enzyme in its sub-subclass.

The subclasses and sub-subclasses are formed according to principles indicated below.

The main divisions and subclasses are:

No.	Class	Type of reaction catalyzed
1	Oxidoreductases	Transfer of electrons (hydride ions or H atoms)
2	Transferases	Group transfer reactions
3	Hydrolases	Hydrolysis reactions (transfer of functional groups to water)
4	Lyases	Addition of groups to double bonds, or formation of double bonds by removal of groups
5	Isomerases	Transfer of groups within molecules to yield isomeric forms
6	Ligases	Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to ATP cleavage