From Models Through Mimics to Artificial Enzymes

Enzymes are the all-purpose catalysts that make the Chemistry of Life run smoothly and efficiently. They do the sorts of things that chemists want to do, under the mildest, "greenest" conditions – in aqueous solution near pH 7, at atmospheric pressure and temperatures close to ambient. They are wonderfully efficient catalysts, capable of handling with ease the most unreactive compounds present in biological systems, and their reactions, where necessary, are completely chemo-, regio- and stereoselective. Small wonder that an understanding of how enzymes work has been an ambition for generations of researchers in a whole range of disciplines, from pure enzymology, through almost all of chemistry to X-ray crystallography.

An understanding of the principles of enzyme catalysis is of far more than academic interest. The industrial use of enzymes is widespread and growing. The food industry has always used the enzymes present in various organisms such as yeasts, but a growing trend is to use isolated enzymes at key stages to improve quality control. Medical applications have become hugely important, both in diagnostic testing and directly in therapeutic applications. And nowhere is the need to understand the fundamental principles more important than in the development of artificial enzymes, which have far-reaching potential in the fine chemicals and pharmaceutical industries. Thus, asymmetric synthesis is a major activity and growth area for organic and pharmaceutical chemistry, and chiral catalysis the most elegant – and most efficient – way of achieving it. Enzymes are chiral catalysts par excellence, and natural (wild-type) or specifically modified enzymes play increasingly important roles.

As proteins, enzymes do, however, have certain practical disadvantages outside their native organisms: they are often denatured inconveniently fast, by changes in pH, heat or solvent, and by surfactants and many other chemicals. And the typical enzyme works best on just one specific substrate, in water, and at concentrations that are inconveniently low for serious synthesis. Hence the interest in developing synthetic "artificial enzymes": which can be more robust, can work in a solvent or solvents of choice; and could in principle be designed to catalyze a particular reaction, rather than a particular reaction of a specific substrate. Last but not least, a major advantage of synthetic systems is that they can in principle be designed to catalyze any reaction of interest, including non-natural reactions, for which no natural enzymes exist. Successful design in this context will inevitably be based on developing enzyme models.

Enzymes are far more than just highly evolved catalysts for specific reactions: they may also have to recognize and respond to molecules other than their specific substrate and product, as part of the control mechanisms of the cell. The evolution of artificial enzymes is at a much more primitive stage, with efficient catalysis the primary, and often the sole, objective. Systems are known that model various other functions, including potential control mechanisms. But to be useful as an industrial catalyst an artificial enzyme has no need of sophisticated built-in feedback control mechanisms or high substrate specificity: a stable molecule that is an efficient catalyst for a key target reaction in a chemical reactor will not be required to select its substrate from many hundreds in the same solution, as enzymes routinely must in the cell. So, a rational design strategy is indeed to consider simply those features of enzymes that are essential for catalytic efficiency.

In these first two chapters we discuss enzyme mechanisms in rather general terms, to identify and define these key features. We then go on to discuss the developing range of enzyme models: by which we mean systems designed to test basic ideas on enzyme mechanism by reproducing specific, key features of enzyme reactions; and attempts to develop them into enzyme-like catalysts. We reserve the term enzyme mimics for the most highly developed enzyme models, which combine successfully more than one of these key features, and catalyze reactions by mechanisms that are demonstrably enzyme-like, involving both binding and catalysis. An enzyme mimic that can do all this, and achieve turnover at a reasonable rate, deserves to be called an artificial enzyme.

1.1 Introduction to Enzyme Chemistry

Enzymes are proteins. The "central dogma" of biological chemistry underlines the pivotal role of enzyme catalysis (and highlights a fascinating problem in biochemical evolution!):

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Enzyme proteins are made up of one (sometimes more than one) polypeptide chain (Figure 1.1), each of which is folded into a flexible, more or less unique active conformation. The preferred 3-dimensional structure is determined by a complex array of physicochemical interactions between side-chains, main-chain amide groups and especially solvent water. Whole books and half a dozen current journals deal specifically with protein chemistry, and the basic ideas are described in many textbooks. So, only those properties of special relevance to catalysis will be introduced in this chapter, and discussed in the necessary detail later in the book. Specific suggestions for further reading are to be found at the end of each chapter.

The easiest way to a broader understanding of the 3-dimensional structures of proteins is to spend time on your computer "playing" with real structures. A good place to start is with the simple-to-use software available at http://www.umass.edu/microbio/rasmol/ or http://www.pymol.org/). While the structure of practically any enzyme of special interest is likely to be one of the many thousands accessible online from the Protein Data Base (http://www.rcsb.org/pdb/).

1.1.1 Why are Enzymes so Big?

Enzymes have evolved to operate under most of the various environments natural to living organisms. The most important of these are the cytoplasm – an aqueous solution containing hundreds of other proteins and small-molecule metabolites – and the surfaces of membranes of various sorts. So enzymes have to be "comfortable" in various operating environments, and "tunable" – to work at different, controlled levels of activity appropriate to the changing requirements of the system. They must also be capable of catalyzing specific reactions of specific substrates at rates (based on values of kcat – see Section 1.2 – typically in the range 1–1000 s-1) high enough to support the immediate demands of the interactive network of local control mechanisms. Substrates range in size from O2 and CO2 to macromolecules, and kcat values between 1–1000 s-1 can represent accelerations of up to 1020 compared with rates of the corresponding...