Enzymes are the (impeccably green) catalysts that make the Chemistry of Life run smoothly and efficiently, and understanding how they work has been a major challenge for Biological Science for many years. Despite tremendous progress our understanding still fails the ultimate, practical test – of designing and making artificial systems with catalytic efficiencies to rival those of natural enzymes. “Artificial enzymes” has been a hot topic for many years, but until now no textbook has been devoted specifically to this subject. From Enzyme Models to Model Enzymes is the first to provide a critical introduction to, and overview of, this exciting area. It is aimed at both students and more senior researchers with interests in the field. The book starts with a systematic overview of the most important properties of natural enzymes, with special emphasis on mechanisms and catalytic efficiency. There follows a summary of the mechanisms involved in the major classes of reaction they catalyze, and of the logical progression from simple mechanistic models for particular reactions to the first, rudimentary model enzymes. Catalytic efficiency is the key criterion for inclusion. A careful analysis of the strengths and limitations of the classical design-based approach to catalysis by enzyme mimics leads on to a critical discussion of recent advances, which combine selection routines with iterative techniques for creating and improving catalysts by biomimetic methods. The meaningful comparison of natural and artificial catalysts requires a quantitative understanding based on the interpretation of kinetic measurements. Key skills in data interpretation are introduced in a guided approach that connects the formal treatment of kinetic measurements with their chemical and biological mechanistic interpretation. This book provides a convenient entry point into the chemistry for the biochemist and molecular biologist, and for the chemist an entrUe into the biological methods that are of rapidly growing importance in this and a number of other topical areas.

Anthony J. Kirby is Professor (Emeritus) of Bioorganic Chemistry at the University of Cambridge. He has over 40 years teaching and research experience in the area and has authored over 300 papers and 3 books. Florian Hollfelder has been lecturing in Biochemistry since 2001. He has a total of 15 years teaching and research experience at Cambridge, Stanford & Harvard and has authored more than 30 papers.

From Enzyme Models to Model Enzymes

By Anthony J. Kirby, Florian Hollfelder

The Royal Society of Chemistry

Copyright © 2009 Anthony J. Kirby and Florian Hollfelder
All rights reserved.
ISBN: 978-0-85404-175-6

Contents

Chapter 1 From Models Through Mimics to Artificial Enzymes, 1,
Chapter 2 Evaluation of Catalytic Efficiency in Enzymes and Enzyme Models, 29,
Chapter 3 Constructing Enzyme Models – Building up Complexity, 42,
Chapter 4 Enzyme Models Classified by Reaction, 61,
Chapter 5 Design vs. Iterative Methods – Mimicking the Way Nature Generates Catalysts, 195,
References, 248,
Subject Index, 266,

CHAPTER 1

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!):

[ILLUSTRATION OMITTED]

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 uncatalyzed reactions at physiological pH in water.

So, an enzyme has to provide a highly sophisticated single-molecule “reaction vessel,” to support such rapid reactions. Not surprisingly, filling and emptying this “reaction vessel” efficiently poses its own problems, because when reactions become very fast, simple diffusion processes can become rate limiting. So, important additional requirements for the active-site “reaction vessel” are rapid substrate binding and – no less important – rapid release of product. (This last is no trivial requirement, considering that the product is typically almost identical to the substrate, give or take the cleavage or formation of a single covalent bond.) Furthermore, most if not all of any water molecules present in the resting active site have to be removed for the duration of the reaction process. All this makes the simple picture of a (static) cavity complementary in structure to the substrate a highly unconvincing proposition. Fischer’s imaginative lock-and-key principle remains a valuable starting point, but an enzyme must provide specific binding complementarity not just to its substrate (or substrates), but to all intermediates and transition states on the reaction pathway.

Thus, the processes involved in catalysis are dynamic, and make complex demands on the enzyme protein. So, we should not be surprised that all this should require a large, sometimes a very large, molecule. An additional if less immediate factor is what might be called “protein bloat”. We are all familiar with the way systems like software applications (or government legislation) that are continually being improved and extended, can grow rapidly in size and complexity over just a small number of years. Enzyme evolution is a great deal slower – but it has been going on for millions of years …

1.1.2 Functional Groups Available to Enzymes

What makes one protein different from another is the sequence of amino-acids, and thus the arrangement of the side-chains Rn (Figure 1.1) in the polypeptide chain. The chemical reactions involved in enzyme catalysis are implemented by the functional groups available on the amino-acid side-chains (backbone amide groups are not usually directly involved). Nine of the 20 naturally occurring amino-acids carry one of six different reactive groups (seven if phenol and alcohol OH groups count as different). These are listed in Table 1.1, which also gives the standard one- and three-letter abbreviations for the amino-acids, and approximate values for the pKas of the side-chain groups. The pKa value tells us how much of each ionic form of a group is present at any given pH, for the amino-acid free in solution. Though it gives only an indication of what might be the situation in the controlled environment of an active site, where in the absence of full solvation pKas can be perturbed, sometimes by as much as 4–5 units. (Qualitatively, this perturbation generally favours neutral species, because ionized forms are stabilized more strongly by hydrogen-bonding solvation.)

The functional groups listed in Table 1.1 are by no means an impressive selection of reagents, by the standards of the present-day organic or inorganic chemist. Nor can they be, because they must operate, and have long-term stability, in water: any strong base, acid or electrophile would immediately be neutralized by the solvent.

1.2 Principles of Catalysis by Enzymes

Most current thinking about enzyme catalysis is based on Pauling’s original suggestion that enzymes work by binding and thus selectively stabilizing the transition states for their reactions. Starting from simple transition-state theory: consider the interaction between two reacting molecules A and B. The essential first step is for the two to come together. In the gas phase this involves a simple collision, but in solution molecules are separated by bulk solvent, and since each has its own solvation shell, making contact is a more complicated business. We can allow for this step by introducing into the reaction pathway the “encounter complex” A · B: without defining it in detail. Once in contact the molecules can undergo multiple “collisions” within the encounter complex before either reacting or diffusing apart. Thus, simple geometrical requirements for reaction, e.g. the directionality of approach of the reacting centres on the separate molecules, are not generally critical. If the chemical reaction is faster than diffusional separation, as is typically the case for many proton-transfer reactions, the diffusion step is rate determining and the reaction is diffusion controlled. (For examples, see Section 4.1.)

A ball-park estimate of the equilibrium constant for the random association of small molecules in aqueous solution is Ka B 0.07 M-1. Making the interaction between A and B “sticky” – i.e. by the various binding interactions of molecular recognition – will increase Ka, and thus, other things being equal, (see Figure 1.4) the overall rate of formation of products. Enzymes work by (i) binding their own particular substrate, usually very specifically from the hundreds available in solution in the cell, (ii) catalyzing a specific reaction of the bound molecule; and (iii) finally releasing the product into solution.

This mode of catalysis – whether by an enzyme or a simpler catalyst – is characterized by “saturation” or “Michaelis–Menten” kinetics: whereby a limiting rate is reached when all catalyst molecules are “busy” – i.e. binding reactant, intermediates or product. The defining equation for Michaelis–Menten kinetics introduces two key parameters, kcat and KM:

rate = [E]0[S]kcat/KM + [S]

In the simplest case, where the chemical step kcat is clearly rate determining (i.e.E.S dissociates faster than it is converted to products), the rate becomes kcat[E]0 at [S] [much greater than] KM, defining kcat as the first-order rate constant for the conversion of E.S to products. And at [S] [much less than] KM (as [S] becomes very small) the rate [right arrow] kcat/KM [E]0[S], defining kcat/KM as the second-order rate constant for the overall reaction. (For further detail see Section 2.3.)

We will use the parameters kcat and KM to characterize catalysis by enzyme models as well as enzymes: because they are familiar, and because they allow direct comparisons with enzyme reactions. We will also use the association constant Ka, in discussions of simple binding equilibria. (This corresponds to 1/KM in the simple Michaelis–Menten mechanism.)

1.2.1 Dependence on pH

The basic mechanistic problem solved by enzyme catalysis is simply illustrated by the schematic pH–rate profiles shown in Figure 1.2. Ionic reactions between nucleophilic and electrophilic reactants, for example most hydrolysis reactions, are typically acid and/or base catalyzed; so slowest (as shown by the minimum in the pH–rate profile I) near pH 7. A sufficiently highly reactive system may react without the need for acid/base catalysis, and show also an uncatalyzed “water reaction” (curve II): which is pH independent over a certain range but still slowest near neutrality. Enzyme reactions, by contrast, are fastest under physiological conditions, often showing bell-shaped pH–rate profiles (curve III), with a rate maximum near pH 7.

1.3 General Acid–Base Catalysis

The reactions at high and low pH (curves I and II of Figure 1.2) are not directly relevant to catalysis by enzymes, which operate of necessity near pH 7, and use catalytic groups that are only weak acids, bases and nucleophiles. The great majority of enzyme-catalyzed reactions are ionic, involving heterolytic bond making and breaking, and thus the creation or neutralization of charge. Under conditions of constant pH this commonly requires the transfer of protons. General acid–base catalysis provides mechanisms for bringing about the necessary proton transfers without involving hydrogen or hydroxide ions, which are present in water at concentrations of the order of 10-7 M under physiological conditions. At pHs near neutrality relatively weak acids and bases can compete effectively with lyonium or lyate species because they can be present in much higher concentrations.

1.3.1 Experimental Evidence

Acid–base catalysis is termed specific if the rate of the reaction concerned depends only on the acidity (pH, etc.) of the medium (as in curve I of Figure 1.2). This is the case if the reaction involves as an intermediate a small amount of the conjugate acid or base of the reactant preformed in a rapid equilibrium process – normal behaviour if the reactant is weakly basic or acidic.

General acid–base catalysis is defined experimentally by the appearance in the rate law of acids and/or bases other than lyonium or lyate ions. Thus, the hydrolysis of enol ethers (Scheme 1.1) is general acid catalyzed: the rate depends on pH, but near neutrality depends also on the concentration of the buffer (HA + A-) used to maintain the pH. Measurements at different buffer ratios show that the catalytic species is the conjugate acid HA. Any “general acid” can be a catalyst: the most reactive will always be the hydronium ion H3O+, which is (by definition) the strongest acid available in aqueous solution.

(Continues…)Excerpted from From Enzyme Models to Model Enzymes by Anthony J. Kirby, Florian Hollfelder. Copyright © 2009 Anthony J. Kirby and Florian Hollfelder. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.