Introduction to Enzyme and Coenzyme Chemistry E-Book

T. D. H. Bugg

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 345

Veröffentlichungsjahr: 2012

Beschreibung

Enzymes are giant macromolecules which catalyse biochemical reactions. They are remarkable in many ways. Their three-dimensional structures are highly complex, yet they are formed by spontaneous folding of a linear polypeptide chain. Their catalytic properties are far more impressive than synthetic catalysts which operate under more extreme conditions. Each enzyme catalyses a single chemical reaction on a particular chemical substrate with very high enantioselectivity and enantiospecificity at rates which approach “catalytic perfection”. Living cells are capable of carrying out a huge repertoire of enzyme-catalysed chemical reactions, some of which have little or no precedent in organic chemistry.

The popular textbook Introduction to Enzyme and Coenzyme Chemistry has been thoroughly updated to include information on the most recent advances in our understanding of enzyme action, with additional recent examples from the literature used to illustrate key points. A major new feature is the inclusion of two-colour figures, and the addition of over 40 new figures of the active sites of enzymes discussed in the text, in order to illustrate the interplay between enzyme structure and function.

This new edition provides a concise but comprehensive account from the perspective of organic chemistry, what enzymes are, how they work, and how they catalyse many of the major classes of enzymatic reactions, and will continue to prove invaluable to both undergraduate and postgraduate students of organic, bio-organic and medicinal chemistry, chemical biology, biochemistry and biotechnology.

Leseprobe

Contents

Cover

Title Page

Copyright

Preface

Representation of Protein Three-Dimensional Structures

Chapter 1: From Jack Beans to Designer Genes

1.1 Introduction

1.2 The discovery of enzymes

1.3 The discovery of coenzymes

1.4 The commercial importance of enzymes in biosynthesis and biotechnology

1.5 The importance of enzymes as targets for drug discovery

Chapter 2: All Enzymes Are Proteins

2.1 Introduction

2.2 The structures of the L-α-amino acids

2.3 The primary structure of polypeptides

2.4 Alignment of amino acid sequences

2.5 Secondary structures found in proteins

2.6 The folded tertiary structure of proteins

2.7 Enzyme structure and function

2.8 Metallo-enzymes

2.9 Membrane-associated enzymes

2.10 Glycoproteins

Chapter 3: Enzymes Are Wonderful Catalysts

3.1 Introduction

3.2 A thermodynamic model of catalysis

3.3 Proximity effects

3.4 The importance of transition state stabilisation

3.5 Acid/base catalysis in enzymatic reactions

3.6 Nucleophilic catalysis in enzymatic reactions

3.7 The use of strain energy in enzyme catalysis

3.8 Desolvation of substrate and active site nucleophiles

3.9 Catalytic perfection

3.10 The involvement of protein dynamics in enzyme catalysis

Chapter 4: Methods for Studying Enzymatic Reactions

4.1 Introduction

4.2 Enzyme purification

4.3 Enzyme kinetics

4.4 The stereochemical course of an enzymatic reaction

4.5 The existence of intermediates in enzymatic reactions

4.6 Analysis of transition states in enzymatic reactions

4.7 Determination of active site catalytic groups

Chapter 5: Hydrolytic and Group Transfer Enzymes

5.1 Introduction

5.2 The peptidases

CASE STUDY: HIV-1 protease

5.3 Esterases and lipases

5.4 Acyl transfer reactions in biosynthesis (coenzyme A)

5.5 Enzymatic phosphoryl transfer reactions

5.6 Adenosine 5′-triphosphate (ATP)

5.7 Enzymatic glycosyl transfer reactions

5.8 Methyl group transfer: use of S-adenosyl methionine and tetrahydrofolate coenzymes for one-carbon transfers

Chapter 6: Enzymatic Redox Chemistry

6.1 Introduction

6.2 Nicotinamide adenine dinucleotide-dependent dehydrogenases

6.3 Flavin-dependent dehydrogenases and oxidases

6.4 Flavin-dependent mono-oxygenases

6.5 CASE STUDY: Glutathione and trypanothione reductases

6.6 Deazaflavins and pterins

6.7 Iron-sulphur clusters

6.8 Metal-dependent mono-oxygenases

6.9 α-Ketoglutarate-dependent dioxygenases

6.10 Non-heme iron-dependent dioxygenases

Chapter 7: Enzymatic Carbon–Carbon Bond Formation

7.1 Introduction

Carbon–carbon bond formation via carbanion equivalents

7.2 Aldolases

CASE STUDY: Fructose 1,6-bisphosphate aldolase

7.3 Claisen enzymes

7.4 Assembly of fatty acids and polyketides

7.5 Carboxylases: Use of biotin

7.6 Ribulose bisphosphate carboxylase/oxygenase (Rubisco)

7.7 Vitamin K-dependent carboxylase

7.8 Thiamine pyrophosphate-dependent enzymes

Carbon–carbon bond formation via carbocation intermediates

7.9 Terpene cyclases

Carbon–carbon formation through radical intermediates

7.10 Phenolic radical couplings

Chapter 8: Enzymatic Addition/Elimination Reactions

8.1 Introduction

8.2 Hydratases and dehydratases

8.3 Ammonia lyases

8.4 Elimination of phosphate and pyrophosphate

8.5 CASE STUDY: 5-Enolpyruvyl shikimate 3-phosphate (EPSP) synthase

Chapter 9: Enzymatic Transformations of Amino Acids

9.1 Introduction

9.2 Pyridoxal 5′-phosphate-dependent reactions at the α-position

9.3 CASE STUDY: Aspartate aminotransferase

9.4 Reactions at the β- and γ-positions of amino acids

9.5 Serine hydroxymethyltransferase

9.6 N-Pyruvoyl-dependent amino acid decarboxylases

9.7 Imines and enamines in alkaloid biosynthesis

Chapter 10: Isomerases

10.1 Introduction

10.2 Cofactor-independent racemases and epimerases

10.3 Keto-enol tautomerases

10.4 Allylic isomerases

10.5 Case Study: Chorismate Mutase

Chapter 11: Radicals in Enzyme Catalysis

11.1 Introduction

11.2 Vitamin B12-dependent rearrangements

11.3 The involvement of protein radicals in enzyme catalysis

11.4 S-adenosyl-methionine-dependent radical reactions

11.5 Biotin synthase and sulphur insertion reactions

11.6 Radical chemistry in DNA repair enzymes

11.7 Oxidised amino acid cofactors and quinoproteins

Chapter 12: Non-Enzymatic Biological Catalysis

12.1 Introduction

12.2 Catalytic RNA

12.3 Catalytic antibodies

12.4 Synthetic enzyme models

Appendix 1: Cahn-Ingold-Prelog Rule for Stereochemical Nomenclature

Appendix 2: Amino Acid Abbreviations

Appendix 3: A Simple Demonstration of Enzyme Catalysis

Appendix 4: Answers to Problems

Index

This edition first published 2012 © 2012 John Wiley & Sons, Ltd

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Library of Congress Cataloging-in-Publication Data

Bugg, T. D. H. Introduction to enzyme and coenzyme chemistry / T. D. H. Bugg. – 3rd ed. p. cm. Includes bibliographical references and index. ISBN 978-1-119-99595-1 (cloth) – ISBN 978-1-119-99594-4 (pbk.) 1. Enzymes. 2. Coenzymes. I. Title. QP601.B955 2012 612′.0151–dc23 2012008853

A catalogue record for this book is available from the British Library.

Cloth ISBN: 9781119995951 Paper ISBN: 9781119995944

Instructors can access PowerPoint files of the illustrations presented within this text, for teaching, at: http://booksupport.wiley.com.

Preface

When I was approached about a 3rd edition of the book, I wanted to enhance the interplay of enzyme active site structure with their catalytic mechanisms and also update the book with some more recent literature examples and topics of current research interest in enzymology. In the 3rd edition I have redrawn the figures showing protein structures using PyMOL software (see Appendix 3). One advantage of PyMOL is that it is easy to prepare high-resolution images, so hopefully the images in the 3rd edition will be a bit sharper than in the 2nd edition. I have used a similar set of examples, but have also added several new examples to supplement the text.

I have also added some new text: in Chapter 3 I have added some new examples of transition state stabilisation and nucleophilic catalysis to Sections 3.4 and 3.6 and added a new Section 3.8 “Desolvation of substrate and active site nucleophiles”, using the example of S. cattleya fluorinase discovered by the group of Professor David O’Hagan. I've also added some new text and references on the role of protein dynamics in enzyme catalysis, which has been a topic of much discussion and debate in recent years. In Chapter 4 I have mentioned the link between hydrogen tunnelling and temperature-independent kinetic isotope effects, also a topic of current interest in enzymology. In Chapter 5 I have mentioned the discovery of a covalent intermediate in the lysozyme reaction by the group of Professor Stephen Withers, and expanded the discussion of glycosyltransferases. In Chapter 11 I have included a new section 11.6 on “Radical chemistry in DNA repair enzymes”, and included a new figure of the Drosophila melanogaster (6-4) photolyase; I'd like to thank Professor Thomas Carell for helpful discussions and information on this enzyme. Finally, in Chapter 12 I have included a figure and discussion about the most ancient catalytic reaction of all: the peptidyl transfer reaction on the ribosome. A great deal of new structural data regarding this reaction has emerged in the last 10 years, although the precise catalytic mechanism is still under debate!

I'd like to thank colleagues, researchers and students at Warwick and elsewhere for their support and encouragement.

T. D. H. BuggUniversity of Warwick January 2012

Representation of Protein Three-Dimensional Structures

In the 3rd edition I have used the program PyMOL to draw representations of protein three-dimensional structures. PyMOL was developed by Warren Lyford DeLano and commercialised by DeLano Scientific LLC. The software is freely available to educational users from the WWW (http://www.pymol.org), can be downloaded with instructions for its use and is supported by a helpful Wiki page (see below). There are several packages available for representation of protein structures (e.g. RASMOL, SwissPDB Viewer) that are freely available and straightforward to use. PyMOL allows the user to save all of the information in the current session, to go back and modify later on and to easily render the protein structure images into high-resolution pictures.

In order to view a protein structure, you must first download the PDB file from the Brookhaven Protein Database, which contains all the data for published X-ray crystal structures and NMR structures of proteins and nucleic acids. I have included the PDB filename for each of the pictures that I have drawn in the figure legend. I recommend that you download a few of these and try viewing each one on a computer screen; you can turn the structure around and get a really good feel for the three-dimensional structure of the protein. You can download the PDB file from http://www.rcsb.org/pdb, or several other web sites.

Once you have downloaded the PDB file (in PDB text format), then you run the PyMOL program and open the PDB structure file to view the structure. You can view the protein backbone in several different ways: as individual atoms (lines or sticks) or protein secondary structure (ribbon or cartoon). In most of the pictures in this edition I have drawn the protein backbone in cartoon format. I have then selected certain catalytic amino acid residues and highlighted them in red, and selected any bound substrate analogues or coenzymes and highlighted them in black or red. In preparing figures for the book I used only black and red, but on a computer screen you can use a wide range of colours and prepare your own multi-colour pictures!

References

W.L. DeLano, “The PyMOL Molecular Graphics System”, DeLano Scientific, San Carlos, CA (2002).

PyMOL home page: http://www.pymol.org

PyMOL Wiki page: http://www.pymolwiki.org

1

From Jack Beans to Designer Genes

1.1 Introduction

Enzymes are giant macromolecules which catalyse biochemical reactions. They are remarkable in many ways. Their three-dimensional structures are highly complex, yet they are formed by spontaneous folding of a linear polypeptide chain. Their catalytic properties are far more impressive than synthetic catalysts which operate under more extreme conditions. Each enzyme catalyses a single chemical reaction on a particular chemical substrate with very high enantioselectivity and enantiospecificity at rates which approach “catalytic perfection”. Living cells are capable of carrying out a huge repertoire of enzyme-catalysed chemical reactions, some of which have little or no precedent in organic chemistry. In this book I shall seek to explain from the perspective of organic chemistry what enzymes are, how they work, and how they catalyse many of the major classes of enzymatic reactions.

1.2 The discovery of enzymes

Historically, biological catalysis has been used by mankind for thousands of years, ever since fermentation was discovered as a process for brewing and bread-making in ancient Egypt. It was not until the 19th century A.D. however that scientists addressed the question of whether the entity responsible for processes such as fermentation was a living species or a chemical substance. In 1897 Eduard Buchner published the observation that cell-free extracts of yeast containing no living cells were able to carry out the fermentation of sugar to alcohol and carbon dioxide. He proposed that a species called “zymase” found in yeast cells was responsible for fermentation. The biochemical pathway involved in fermentation was subsequently elucidated by Embden and Meyerhof – the first pathway to be discovered.

The exquisite selectivity of enzyme catalysis was recognised as early as 1894 by Emil Fischer, who demonstrated that the enzyme which hydrolyses sucrose, which he called “invertin”, acts only upon α-D-glucosides, whereas a second enzyme “emulsin” acts only upon β-D-glucosides. He deduced that these two enzymes must consist of “asymmetrically built molecules”, and that “the enzyme and glucoside must fit each other like a lock and key to be able to exert a chemical influence upon each other”. Fischer's “lock and key” hypothesis remained a powerful metaphor of enzyme action for many years. The crystallisation in 1926 of the enzyme urease (see Figure 1.1) from Jack beans by Sumner proved beyond doubt that biological catalysis was carried out by a chemical substance.

Figure 1.1 Urease.

The recognition that biological catalysis is mediated by enzymes heralded the growth of biochemistry as a subject, and the elucidation of the metabolic pathways catalysed by enzymes. Each reaction taking place on a biochemical pathway is catalysed by a specific enzyme. Without enzyme catalysis the uncatalysed chemical process would be too slow to sustain life. Enzymes catalyse reactions involved in all facets of cellular life: metabolism (the production of cellular building blocks and energy from food sources); biosynthesis (how cells make new molecules); detoxification (the breakdown of toxic foreign chemicals); and information storage (the processing of deoxyribonucleic acids).

In any given cell there are present several thousand different enzymes, each catalysing its specific reaction. How does a cell know when it needs a particular enzyme? The production of enzymes, as we shall see in Chapter 2, is controlled by a cell's DNA, both in terms of the specific structure of a particular enzyme and the amount which is produced. Thus different cells in the same organism have the ability to produce different types of enzymes and to produce them in differing amounts according to the cell's requirements.

Since the majority of the biochemical reactions involved in cellular life are common to all organisms, a given enzyme will usually be found in many or all organisms, albeit in different forms and amounts. By looking closely at the structures of enzymes from different organisms which catalyse the same reaction, we can in many cases see similarities between them. These similarities are due to the evolution and differentiation of species by natural selection. So by examining closely the similarities and differences of an enzyme from different species we can trace the course of molecular evolution, as well as learning about the structure and function of the enzyme itself.

Recent developments in biochemistry, molecular biology and X-ray crystallography now allow a far more detailed insight into how enzymes work at a molecular level. We now have the ability to determine the amino acid sequence of enzymes with relative ease, whilst the technology for solving the three-dimensional structure of enzymes is developing apace. We also have the ability to analyse their three-dimensional structures using molecular modelling and then to change the enzyme structure rationally using site-directed mutagenesis. We are now starting to enter the realms of enzyme engineering, where by rational design we can modify the genes encoding specific enzymes, creating the “designer genes” of the title. These modified enzymes could in future perhaps be used to catalyse new types of chemical reactions, or via gene therapy to correct genetic defects in cellular enzymes which would otherwise lead to human diseases.

1.3 The discovery of coenzymes

At the same time as the discovery of enzymes in the late 19th and early 20th centuries, a class of biologically important small molecules was being discovered which had remarkable properties to cure certain dietary disorders. These molecules were christened the vitamins, a corruption of the phrase “vital amines” used to describe their dietary importance (several of the first-discovered vitamins were amines, but this is not true of all the vitamins). The vitamins were later found to have very important cellular roles, shown in Table 1.1.

Table 1.1 The vitamins.

The first demonstration of the importance of vitamins in the human diet took place in 1753. A Scottish naval physician James Lind found that the disease scurvy, prevalent amongst mariners at that time, could be avoided by deliberately including green vegetables or citrus fruits in the sailors’ diets. This discovery was used by Captain James Cook to maintain the good health of his crew during his voyages of exploration in 1768–1776. The active ingredient was elucidated much later as vitamin C, ascorbic acid.

The first vitamin to be identified as a chemical substance was thiamine, lack of which causes the limb paralysis beriberi. This nutritional deficiency was first identified in the Japanese Navy in the late 19th century. The incidence of beriberi in sailors was connected with their diet of polished rice by Admiral Takaki, who eliminated the ailment in 1885 by improvement of the sailors’ diet. Subsequent investigations by Eijkman identified a substance present in rice husks able to cure beriberi. This vitamin was subsequently shown to be an essential “cofactor” in cellular decarboxylation reactions, as we shall see in Chapter 7.

Over a number of years the family of vitamins shown in Table 1.1 was identified and their chemical structures elucidated. Some like vitamin C have simple structures, whilst others like vitamin B12 have very complex structures. It has taken much longer to elucidate the molecular details of their biochemical mode of action. Many of the vitamins are in fact co-enzymes: small organic co-factors which are used by certain types of enzyme in order to carry out particular classes of reaction. Table 1.1 gives a list of the co-enzymes that we are going to encounter in this book.

1.4 The commercial importance of enzymes in biosynthesis and biotechnology

Many plants and micro-organisms contain natural products which possess potent biological activities. The isolation of these natural products has led to the discovery of many biologically active compounds such as quinine, morphine, and penicillin (see Figure 1.2) which have been fundamental to the development of modern medicine.

Figure 1.2 Structures of quinine, morphine, penicillin.

The process of natural product discovery continues today, with the recent identification of important compounds such as cyclosporin A, a potent immunosuppressant which has dramatically reduced the rejection rate in organ transplant operations; and taxol, an extremely potent anticancer drug isolated from yew bark (see Figure 1.3).

Figure 1.3 Structures of CsA, taxol.

Many of these natural products are structurally so complex that it is not feasible to synthesise them in the laboratory at an affordable price. Nature, however, is able to bio-synthesise these molecules with apparent ease using enzyme-catalysed biosynthetic pathways. Hence there is considerable interest in elucidating the biosynthetic pathways for important natural products and using the enzymes to produce natural products in vitro. One example of this is the industrial production of semi-synthetic penicillins using a naturally occurring enzyme penicillin acylase (see Figure 1.4). Penicillin G which is obtained from growing Penicillium mould has certain clinical disadvantages; enzymatic deacylation and chemical re-acylation give a whole range of “semi-synthetic” penicillins which are clinically more useful.

The use of enzyme catalysis for commercial applications is an exciting area of the biotechnology industry. One important application that we shall encounter is the use of enzymes in asymmetric organic synthesis. Since enzymes are highly efficient catalysts that work under mild conditions and are enantiospecific, they can in many cases be used on a practical scale to resolve racemic mixtures of chemicals into their optically active components. This is becoming increasingly important in drug synthesis, since one enantiomer of a drug usually has very different biological properties from the other. The unwanted enantiomer might have detrimental side-effects, as in the case of thalidomide, where one enantiomer of the drug was useful in relieving morning sickness in pregnant women, but the other enantiomer caused serious deformities in the newborn child when the racemic drug was administered.

1.5 The importance of enzymes as targets for drug discovery

If there is an essential enzyme found uniquely in a certain class of organisms or cell type, then a selective inhibitor of that enzyme could be used for selective toxicity against that organism or cell type. Similarly, if there is a significant difference between a particular enzyme found in bacteria as compared with the same enzyme in humans, then a selective inhibitor could be developed for the bacterial enzyme. If this inhibitor did not inhibit the human enzyme, then it could be used as an antibacterial agent. Thus, enzyme inhibition is a basis for drug discovery.

This principle has been used for the development of a range of pharmaceutical and agrochemical agents (Table 1.2) – we shall see examples of important enzyme targets later in the book. In many cases resistance to these agents has emerged due to mutation in the structures of the enzyme targets. This has provided a further incentive to study the three-dimensional structures of enzyme targets, and has led to the development of powerful molecular modelling software for analysis of enzyme structure and de novo design of enzyme inhibitors.

Table 1.2 Commercial applications of enzyme inhibitors.

Antibacterial agentsPenicillins and cephalosporins inactivate the transpeptidase enzyme which normally makes cross-links in the bacterial cell wall (peptidoglycan), leading to weakened cell walls and eventual cell lysis. Streptomycin and kanamycin inhibit protein synthesis on bacterial ribosomes, whereas mammalian ribosomes are less affected.Antifungal agentsKetoconazole inhibits lanosterol 14α-demethylase, an enzyme involved in the biosynthesis of an essential steroid component of fungal cell membranes. Nikkomycin inhibits chitin synthase, an enzyme involved in making the chitin cell walls of fungi.Antiviral agentsAZT inhibits the reverse transcriptase enzyme required by the HIV virus in order to replicate its own DNA.InsecticidesOrganophosphorus compounds such as dimethoate derive their lethal activity from the inhibition of the insect enzyme acetylcholinesterase involved in the transmission of nerve impulses.HerbicidesGlyphosate inhibits the enzyme EPSP synthase which is involved in the biosynthesis of the essential amino acids phenylalanine, tyrosine and tryptophan (see Chapter 8.5).

Figure 1.4 Penicillin acylase.

The next two chapters are “theory” chapters on enzyme structure and enzyme catalysis, followed by a “practical” chapter on methods used to study enzymatic reactions. Chapters 5–11 cover each of the major classes of enzymatic reactions, noting each of the coenzymes used for enzymatic reactions. Finally there is a brief introduction in Chapter 12 to other types of biological catalysis. In cases where discussion is brief the interested reader will find references to further reading at the end of each chapter.

2

All Enzymes Are Proteins

2.1 Introduction

Enzymes are giant molecules. Their molecular weight varies from 5,000 to 5,000,000 Da, with typical values in the range 20,000–100,000 Da. At first sight this size suggests a bewildering complexity of structure, yet we shall see that enzymes are structurally assembled in a small number of steps in a fairly simple way.