Enzymes, their substrates and other metabolites

Enzymes, their substrates and other metabolites. 289 Metabolite analogues definition, derivation, and mode of action. 295 History of metabolite antagonism prior to 1940. 302 The folic acid antagonists. 303 

The process of growth and division involves cells in ceaseless chemical activity. For the most part this activity takes the form of chemical reactions between enzymes and substrates, whereby the enzymes remain unchanged, and the substrates are transformed into other metabolites by the breaking or making of covalent bonds. Even the organic coenzymes may undergo 

Until recently, the conventional purification of enzymes, e.g. by electrophoresis, was slow and the yields poor. The recent introduction of affinity chromatography enables an enzyme to be obtained pure and in high yield. In this technique, a specific inhibitor of the enzyme is attached, covalently and by a flexible link such as-CHg.CHg-, to a polymeric adsorbant. The enzyme collects on the inhibitor, from which it can usually be freed by a change in pH (Cuatrecasas, Wilcheck and Anfinsen, 1968). 

The first enzyme to be completely synthesized was ribonuclease. This was done both in solution, by the classical chemical methods (Hirschmann et al. 1969), and by Merrifield s solid-support technique (Gutte and Merri-field, 1969). 

The most successful method for disclosing enzyme structure is the X-ray diffraction study of a suitable crystal. For definitive results, a resolution of 2 A is eventually attempted, but this is extremely time-consuming, even with computational aid. The special value of X-ray diffraction is that it reveals the tertiary folding of the enzyme molecule, so that it can be seen which aminoacids are physically close to one another in the folded state. It is known that the active site of an enzyme is usually composed of two or three aminoacid residues that would lie far apart if the chain were extended. For example, the action of the enzyme lysozyme (from white of egg) on its substrate in the bacterial cell wall (murein Fig. 5.3) requires the proximity of residues 35 and 52 (glutamic and aspartic acid, respectively) from two strands of the enzyme (Phillips, 1966). Lysozyme has predominantly a hydrophobic interior and hydrophilic exterior in its tertiary structure, an arrangement quite common in enzymes. 

---