The Chemical Reactivity Of Enzymes

An enzyme contains one or more active sites, at which the reaction occurs. The substrate, the substance that is being altered, becomes attached to this site in some way. A co-enzyme, if one is involved, is also attached to, or held close to the active site.

"An enzyme first binds its substrate in a particular orientation by using a variety of weak binding forces (hydrogen bonding, electrostatic attraction, dipole-dipole interaction, hydrophobic attraction, and so on), and then uses a variety of strategically placed functional groups and controlled conformational changes to induce reaction between them."

J. W. Cornforth, 1984, see Further Reading

Cornforth has done much of the work in understanding the stereochemistry of many biosynthetic reactions and was awarded the Nobel Prize for Chemistry (with V. Prelog) for this in 1975. For the biochemistry of enzymes the reader is directed to T. Palmer, Understanding enzymes 3rd edtion, 1991, Ellis Horwood, Chichester, and for a detailed treatment of enzyme and co-enzyme reaction mechanisms, T. Bugg, An introduction to enzyme and co-enzyme chemistry 1997, Blackwell Scientific, Oxford.

2.1.1 Lysozyme

Lysozyme has often been chosen as a simple example of how an enzyme works. It is said that Alexander Fleming (who later discovered penicillin) when he had a cold, at one time let the drips from his nose fall onto a bacterial colony on a Petri dish. Rather than throw it away, he kept it to see what would happen. He discovered that his nasal discharge inhibited the growth of the bacterium and this led to the discovery of the mildly antibiotic substance lysozyme in tears. He gave it this name because it is an enzyme that caused bacterial lysis. Later lysozyme was found in other body fluids, and elsewhere, but particularly in the white of egg. Lysozyme acts on a group of bacteria that have a cross-linked polysaccharide on their cell surfaces. Lysozyme cuts up the polysaccharide, making the bacterial cell wall very fragile.

Lysozyme has a relatively small molecule for a protein, with 129 amino-acid residues linked in a single protein chain, and a molecular mass of 13,930. It was the first enzyme to have its total structure determined by X-ray analysis (1965), and to have its active site discovered. The protein chain of lysozyme is twisted and folded into a shape like a ball with a cleft down one side. The polysaccharide network on the surface of the bacteria fits into the cleft. The structure of the polysaccharide (the substrate) is shown in Figure 2.1 and the schematic structure of the enzyme molecule is shown in Figure 2.2 with the polysaccharide adsorbed on to the active site ready to be cleaved. Six rings of the

Figure 2.1 The polysaccharide molecule found in the walls of certain bacterial cells is the substrate broken by the lysozyme molecule. The polysaccharide consists of alternating residues of two kinds of amino sugar: N-acetylglucosamine and N-acetylmuramic acid. In the portion of polysaccharide chain shown here A, C and E are N-acetylglucosamine residues; B, D and Fare N-acetylmuramic acid residues. Ring D is distorted when adsorbed on the enzyme. The position attacked is indicated by the arrows

Figure 2.2 The amino-acid chain of lysozyme (a) is folded so it roughly forms a ball, with a cleft down one side, into which the polysaccharide chain of the bacterium fits. In (b) is shown how the aspartic acid 52 and glutamic acid 35 work together to break the sugar chain

Figure 2.1 The polysaccharide molecule found in the walls of certain bacterial cells is the substrate broken by the lysozyme molecule. The polysaccharide consists of alternating residues of two kinds of amino sugar: N-acetylglucosamine and N-acetylmuramic acid. In the portion of polysaccharide chain shown here A, C and E are N-acetylglucosamine residues; B, D and Fare N-acetylmuramic acid residues. Ring D is distorted when adsorbed on the enzyme. The position attacked is indicated by the arrows

Jn UN 1 OH

HOOC ^ HOOC^ HOOC

Jn UN 1 OH

HOOC ^ HOOC^ HOOC

(a)

released released

Figure 2.2 The amino-acid chain of lysozyme (a) is folded so it roughly forms a ball, with a cleft down one side, into which the polysaccharide chain of the bacterium fits. In (b) is shown how the aspartic acid 52 and glutamic acid 35 work together to break the sugar chain released released polysaccharide fit into the cleft of the lysozyme molecule, and are held firmly in position by hydrogen bonds and other interactions (see the quotation from Cornforth, above). Ring D is held in a flattened conformation, so the bond to ring E is strained and prepared for reaction. Adjacent to it are two carboxylic acid groups; that of aspartic acid 52 is in polar surroundings and is in the ionized form; that of glutamic acid 35 is in non-polar surroundings and is therefore in the un-ionized form. These two groups and a water molecule complete the reaction as shown in Figure 2.2.

2.1.2 Carboxypeptidase

In the example of lysozyme, the catalytic effect is entirely due to the protein. In many enzymes there is a prosthetic group that often contains a metal ion bound to the protein, as in the example of carboxypeptidase, which contains a zinc atom in the active site (Figure 2.3), that takes part in the reaction. There are at least four carboxypepti-dases which differ in the particular amino-acid linkages they are able

Figure 2.3 The carboxylic acid end of a protein sitting in the active site, a pocket in the enzyme carboxypeptidase, showing how it is held in place by bonding to the Zn2+ atom and various amino acids. The probable mechanism, based on the hydrolysis of a known peptide, is shown, with the water molecule used for the hydrolysis shown in bold type. Once the amino-acid is cleaved, it can diffuse away and the protein moves up into the pocket for the next amino-acid to be cleaved in the same way

P"/ H3N+-Arg145

to hydrolyze. Carboxypeptidase hydrolyzes amino-acids from the carboxyl end of proteins, one by one. Some 400 enzymes are known that contain zinc atoms. Other common metals in enzymes are Fe, Mg, Ca and Mn.

A more advanced picture of enzyme action in biosynthesis is given in Chapter 3 when discussing desaturase enzymes.

2.1.3 Cytochromes

There is in all living cells a family of enzymes known as cytochrome P450 oxygenases. They contain a haem group (Figure 2.4) attached covalently, and have an absorption maximum at 450 nm in the violet region of the spectrum, and are coloured yellow, hence their names. They are important in the oxidizing of alkanes to alcohols, alkenes to epoxides, in the transformation of sterols (see Chapter 7) and for introducing an OH group into aromatic rings. They are also important for the detoxifying of many ingested substances, whether they be from plants or animals, or are synthetic substances, like pesticides or environmental pollutants. Cytochromes activate molecular oxygen, which attacks the substrate of whatever kind, as in Figure 2.5. Notice that the Fe3+ atom must be re-oxidized to Fe4+ before the enzyme can be used again. It accepts another molecule of 02, splits off OH" and is restored to Fe4+-0- for re-use. Other substances that contain haem are haemoglobin, other cytochromes and catalase.

Figure 2.4 The structure of haem. The tetrapyrrole without the iron atom is known as protoporphyrin IX (see also Chapter 8)
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