Enzymes facilitate life via a plethora of reactions in living organisms. Not only do they sustain life – they are also involved in a myriad of processes that affect our everyday lives. These include applications in medicine, household detergents, fine chemical synthesis, the food industry, bioelectronics and the degradation of chemical waste. Since the discovery of enzymes just over a century ago, we have witnessed an explosion in our understanding of enzyme catalysis, leading to a more detailed appreciation of how they work. Over many years, much effort has been expended in the quest to create enzymes for specific biotechnological roles. Prior to the early 1980s, the only methods available for changing enzyme structure were those of chemical modification of functional groups (so-called ‘forced evolution’). The genetic engineering revolution has provided tools for dissecting enzyme structure and enabling design of novel function. Chemical methods have now been surpassed by knowledge-based (i.e. rational) sitedirected mutagenesis and the grafting of biological function into existing enzyme molecules (so-called ‘retrofitting’). More recently, gene-shuffling techniques have been used to generate novel enzymes. Rational redesign of enzymes is a logical approach to producing better enzymes. However, with a few notable exceptions, rational approaches have been generally unsuccessful, reiterating our poor level of understanding of how enzymes work. This has led to a more ‘shot-gun’ approach to redesign, involving random mutagenesis – producing modest success, but dependent on being able to ‘pull out’ an improved enzyme by ‘fishing’ in a very large collection of randomly modified enzymes. However, development of a suitable test (i.e. producing the correct ‘bait’) to identify an improved enzyme is intrinsically very difficult. Therefore the rational approach, although generally unsuccessful, cannot be ignored.
Enzymes are large biological molecules – usually proteins – that speed up chemical reactions. Molecules that speed up chemical reactions, but are unchanged afterwards, are known as catalysts. The substances that enzymes act on are known as substrates. Enzymes exhibit remarkable specificity for their substrate molecules, and can approach ‘catalytic perfection’. A popular approach to modelling catalysis has been to visualise an energy barrier that must be surmounted to proceed from reactants to products. The greater the height of this energy barrier, the slower the rate of reaction. Enzymes (like other catalysts) reduce the energy required to pass over this barrier, thereby increasing reaction rate. The structure of the reactant at the top of the barrier is energetically unstable, and is known as the ‘transition state’. The energy required to pass over the barrier is the so-called ‘activation energy’ – the barrier is surmounted by thermal excitation of the substrate. This classical over-the-barrier treatment – known as transition state theory – has been used to picture enzymecatalysed reactions over the past 50 years. However, recent developments indicate that this ‘textbook’ illustration is fundamentally flawed (at least in some circumstances).
