eureka eureka

Biochemistry & Metabolism Biochemistry Biochemistry &

eureka Metabolism Andrew Davison, Anna Milan, Suzannah Phillips, Lakshminarayan Ranganath medicine made clear Core science and clinical cases in one book

eureka – an innovative series for students that fully integrates core science, clinical medicine and surgery. With its engaging and authoritative text, featuring insightful clinical cases, graphic narratives, SBAs and a wealth of other learning tools, Eureka has everything students need to succeed in medicine and pass their exams.

> First principles chapter explains key > Clinical cases teach you to think like

concepts and mechanisms of a doctor Davison, Milan, Phillips, Ranganath biochemistry > Graphic narratives bring cases to life > Systems-based chapters describe the > Starter questions stimulate curiosity processes that underpin normal and learning function > Clinical SBAs chapter helps you revise and pass your exams

Series Editors: Janine Henderson, David Oliveira, Stephen Parker

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EurekaBiochem_COVER_AW.indd 1 11/08/2015 13:42 eureka

Biochemistry & Metabolism

eureka

Biochemistry & Metabolism

Andrew Davison BSc (Hons) MSc CSci Suzannah Phillips BSc (Hons) MSc PhD EuSpLM FRCPath Principal Clinical Biochemist and Principal Clinical Biochemist and Honorary Lecturer in Clinical Honorary Lecturer in Clinical Biochemistry Biochemistry Department of Clinical Biochemistry Department of Clinical Biochemistry Liverpool Clinical Laboratories Liverpool Clinical Laboratories Royal Liverpool and Broadgreen Royal Liverpool and Broadgreen University Hospitals NHS Trust University Hospitals NHS Trust Liverpool, UK Liverpool, UK Lakshminarayan Ranganath MD Anna Milan BSc (Hons) MSc PhD MBBS MSc PhD CSci FRCP FRCPath EuSpLM FRCPath Professor and Consultant in Chemical Principal Clinical Biochemist and Pathology Honorary Lecturer in Clinical Department of Clinical Biochemistry Biochemistry Liverpool Clinical Laboratories Department of Clinical Biochemistry Royal Liverpool and Broadgreen Liverpool Clinical Laboratories University Hospitals NHS Trust Royal Liverpool and Broadgreen Liverpool, UK University Hospitals NHS Trust Liverpool, UK

Series Editors

Janine Henderson MRCPsych Stephen Parker BSc MS DipMedEd MClinEd FRCS MB BS Programme Director Consultant Breast and General Hull York Medical School Paediatric Surgeon York, UK St Mary’s Hospital Newport, UK David Oliveira PhD FRCP Professor of Renal Medicine St George’s, University of London London, UK

London • Philadelphia • New Delhi • Panama City © 2015 JP Medical Ltd. Published by JP Medical Ltd, 83 Victoria Street, London, SW1H 0HW, UK First reprint 2015 Tel: +44 (0)20 3170 8910 Fax: +44 (0)20 3008 6180 Email: [email protected] Web: www.jpmedpub.com

The rights of Andrew Davison, Anna Milan, Suzannah Phillips and Lakshminarayan Ranganath to be identified as the authors of this work have been asserted by them in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission in writing of the publishers. Permissions may be sought directly from JP Medical Ltd at the address printed above. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. Medical knowledge and practice change constantly. This book is designed to provide accurate, authoritative information about the subject matter in question. However readers are advised to check the most current information available on procedures included and check information from the manufacturer of each product to be administered, to verify the recommended dose, formula, method and duration of administration, adverse effects and contraindications. It is the responsibility of the practitioner to take all appropriate safety precautions. Neither the publisher nor the authors assume any liability for any injury and/or damage to persons or property arising from or related to use of material in this book. This book is sold on the understanding that the publisher is not engaged in providing professional medical services. If such advice or services are required, the services of a competent medical professional should be sought. Every effort has been made where necessary to contact holders of copyright to obtain permission to reproduce copyright material. If any have been inadvertently overlooked, the publisher will be pleased to make the necessary arrangements at the first opportunity.

ISBN: 978-1-907816-83-3 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library

Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress

Publisher: Richard Furn Development Editors: Thomas Fletcher, Paul Mayhew, Alison Whitehouse Editorial Assistants: Sophie Woolven, Katie Pattullo Copy Editor: Kim Howell Graphic narratives; James Pollitt Cover design: Forbes Design Interior design: Designers Collective Ltd eureka Series Editors’ Foreword

Today’s medical students need to know a great deal to be effective as tomorrow’s doctors. This knowledge includes core science and clinical skills, from understanding biochemical pathways to communicating with patients. Modern medical school curricula integrate this teaching, thereby emphasising how learning in one area can support and reinforce another. At the same time students must acquire sound clinical reasoning skills, working with complex information to understand each individual’s unique medical problems. The Eureka series is designed to cover all aspects of today’s medical curricula and reinforce this integrated approach. Each book can be used from first year through to qualification. Core biomedical principles are introduced but given relevant clinical context: the authors have always asked themselves, ‘why does the aspiring clinician need to know this’? Each clinical title in the series is grounded in the relevant core science, which is introduced at the start of each book. Each core science title integrates and emphasises clinical relevance throughout. Medical and surgical approaches are included to provide a complete and integrated view of the patient management options available to the clinician. Clinical insights highlight key facts and principles drawn from medical practice. Cases featuring unique graphic narratives are presented with clear explanations that show how experienced clinicians think, enabling students to develop their own clinical reasoning and decision making. Clinical SBAs help with exam revision while Starter questions are a unique learning tool designed to stimulate interest in the subject. Having biomedical principles and clinical applications together in one book will make their connections more explicit and easier to remember. Alongside repeated exposure to patients and practice of clinical and communication skills, we hope Eureka will equip medical students for a lifetime of successful clinical practice.

Janine Henderson, David Oliveira, Stephen Parker vi

About the Series Editors

Janine Henderson is the MB BS undergraduate Programme Director at Hull York Medical School (HYMS). After medical school at the University of Oxford and clinical training in psychiatry, she combined her work as a consultant with postgraduate teaching roles, moving to the new Hull York Medical School in 2004. She has a particular interest in modern educational methods, curriculum design and clinical reasoning. David Oliveira is Professor of Renal Medicine at St George’s, University of London (SGUL), where he served as the MBBS Course Director between 2007 and 2013. Having trained at Cambridge University and the Westminster Hospital he obtained a PhD in cellular immunology and worked as a renal physician before being appointed as Foundation Chair of Renal Medicine at SGUL. Stephen Parker is a Consultant Breast and General Paediatric Surgeon at St Mary’s Hospital, Isle of Wight. He trained at St George’s, University of London, and after service in the Royal Navy was appointed as Consultant Surgeon at University Hospital Coventry. He has a particular interest in e-learning and the use of multimedia platforms in medical education.

About the Authors

Andrew Davison is a Principal Clinical Biochemist with a special interest in phaeochromocytoma and one of the co-leads for the MSc course in Clinical Biochemistry at the University of Manchester. He has a keen interest in teaching undergraduate and postgraduate students about the science behind clinical medicine. Anna Milan is a Principal Clinical Biochemist with a keen interest in bone markers, vitamins, mineralised tissue biochemistry and alkaptonuria. She is an Honorary Lecturer at the University of Liverpool and also lectures on the MSc course in Clinical Biochemistry at the University of Manchester. She has over 15 years’ experience in teaching at all levels, in clinical and non-clinical settings, through lectures and problem-based learning examples. Suzannah Phillips is a Principal Clinical Biochemist at the Royal Liverpool University Hospital. She has over 10 years’ experience in teaching both undergraduate and postgraduate students and coordinates undergraduate medical teaching in clinical biochemistry. She also lectures on the MSc course in Clinical Biochemistry at the University of Manchester. Lakshminarayan Ranganath is a Consultant Chemical Pathologist at the Royal Liverpool University Hospital and Clinical Director of the National Alkaptonuria Centre. He has extensive experience in teaching students, medical and non-medical trainees. vii

Preface

Knowledge of biochemistry is vital to the practise of clinical medicine, providing an understanding of disease processes at the molecular level. It is essential that clinicians and scientists have a firm grasp of the science that underpins disease, in order to select the appropriate treatment and investigations for their patients. Eureka Biochemistry & Metabolism covers the fundamental building blocks of life, from basic metabolism to the investigation of disease. Chapter 1 provides an introduction to cellular structure and function, biochemical reactions and body fuels. Chapters 2-6 build on these themes and are enhanced by clinical cases; these highlight the importance of the core science and emphasise its relevance to real life and clinical medicine. Chapters 7 and 8 describe fluid and electrolyte balance and nutrition; like all the other chapters they include clinical insights to help clinicians make informed decisions around patient care. Lastly, chapter 9 provides an invaluable revision aid for undergraduate students in the form of clinical SBAs. Throughout Eureka Biochemistry & Metabolism, we include diagrams and unique graphic narratives to help students understand key concepts. We have made every effort to carefully explain the knowledge you will need to succeed in your exams and become a successful doctor. We hope you enjoy the book and find it useful.

Andrew Davison, Anna Milan, Suzannah Phillips, Lakshminarayan Ranganath April 2015 viii

Contents

Series Editors’ Foreword v Protein structure and function 83 About the Series Editors vi Protein turnover 87 About the Authors vi Preface vii Chapter 4 Carbohydrates Glossary x Introduction 93 Acknowledgements xii Case 3 Collapsed and unresponsive 94

Chemical energy 96 Chapter 1 First principles Glucose breakdown 102 Introduction 1 The pentose phosphate pathway 107 Overview 1 The citric acid cycle 110 Cell structure 3 Oxidative phosphorylation 112 Cell membranes and the transport Glycogen 116 of molecules and ions 8 Gluconeogenesis 119 Signalling pathways 14 Other fuels: fructose and galactose 121 Enzymes and cofactors 20 Biochemical bonds and reactions 26 Chapter 5 Lipids Body fuels 32 Introduction 125 Chapter 2 Nucleic acids Case 4 Tiredness and tingling fingers 126 Lipid structures 128 Introduction 43 Lipolysis 129 Case 1 Pain and swelling in a big toe 44 Lipogenesis 135 Nucleotides 46 Ketone body metabolism 140 Nucleotide synthesis 49 Cholesterol metabolism 141 Nucleotide degradation 54 Drugs targeting nucleotide metabolism 56 Chapter 6 Haemoglobin Nucleic acid structure 57 metabolism Nucleic acid synthesis 60 Introduction 153 Mutations 65 Case 5 Chest pain 154

Chapter 3 Proteins Blood cells 156 Haemoglobin structure 159 Introduction 69 Haemoglobin synthesis 160 Case 2 Seizure and lethargy in a baby 70 Haemoglobin breakdown 163 Amino acids 72 Iron homeostasis 165 Protein synthesis 79 ix Contents

Haemoglobin function 170 Chapter 8 Nutrition Disorders of haemoglobin 174 Introduction 203 Case 7 Yellow skin and diarrhoea 205 Chapter 7 Body fluid homeostasis Energy balance 207 Vitamins 209 Introduction 179 Minerals and trace elements 223 Case 6 Injuries sustained in a traffic collision 180 Nutrition and disease 227 pH 181 Systems that maintain acid–base Chapter 9 Self-assessment homeostasis 182 SBA questions 237 Acid–base balance 187 SBA answers 245 Maintenance of fluid balance 191

Maintenance of electrolyte balance 194 Index 253 x

Glossary

A site aminoacyl site DNA deoxyribonucleic acid ABCA1 ATP-binding cassette transporter A1 dNTP deoxyribonucleoside triphosphate ABCG1 ATP-binding cassette transporter G1 dTMP deoxythymidine monophosphate ACE angiotensin-converting enzyme dTTP deoxythymidine triphosphate ADP adenosine diphosphate dUDP deoxyuridine diphosphate ADH antidiuretic hormone dUMP deoxyuridine monophosphate AMP adenine monophosphate dUTP deoxyuridine triphosphate ANP atrial natriuretic peptide APRT adenine phosphoribosyltransferase E enzyme ATP adenosine triphosphate EDTA ethylenediaminetetra-acetic acid AVP arginine vasopressin F ferroportin 1 BMD bone mineral density FAD+ flavin adenine dinucleotide (oxidised BNP brain natriuretic peptide form)

FADH2 flavin adenine dinucleotide (reduced form) C:D ratio of number of carbon 2+ atoms:double bonds Fe oxidising iron 3+ cAMP cyclic adenosine monophosphate Fe ferric iron CDP cytidine diphosphate FMN flavin mononucleotide (reduced form) CE cholesterol ester CHOL cholesterol G Gibbs free energy CMP cytidine monophosphate GDP guanosine diphosphate CoA coenzyme A GLUT glucose transporter CoASH coenzyme A (reduced form) GMP guanine monophosphate CTP cytidine triphosphate GP general practitioner cyt cytochrome G-protein guanine nucleotide–binding protein GTP guanosine triphosphate

dADP deoxyadenosine diphosphate DAG diacylglycerol H enthalpy dATP deoxyadenosine triphosphate H hephaestin dCDP deoxycytidine diphosphate Hb haemoglobin dCTP deoxycytidine triphosphate HbA adult haemoglobin HbA DEXA dual-energy X-ray absorptiometry 2 haemoglobin A2 dGDP deoxyguanosine diphosphate HbC haemoglobin C dGTP deoxyguanosine triphosphate HbD haemoglobin D HbD DHF dihydrofolate punjab haemoglobin Punjab DHFR dihydrofolate reductase HbF fetal haemoglobin Hb DMT-1 divalent metal iron transporter-1 lepore haemoglobin Lepore xi Glossary

HbM haemoglobin M PL phospholipid HbS sickle haemoglobin pre-mRNA precursor messenger ribonucleic acid HbSS sickle cell anaemia pre-RNA precursor ribonucleic acid HDL high-density lipoprotein pre-rRNA precursor ribosomal ribonucleic acid HGPRT hypoxanthine–guanine pre-tRNA precursor transfer ribonucleic acid phosphoribosyltransferase PRPP 5-phosphoribosyl 1-pyrophosphate HH hereditary haemochromatosis

HMG CoA 3-hydroxy-3-methylglutaryl CoA RANK receptor-activated nuclear kappa RANKL receptor-activated nuclear kappa-B IDL intermediate-density lipoprotein ligand IMP inosine monophosphate RNA ribonucleic acid rRNA IP3 inositol 1,4,5-triphosphate ribosomal ribonucleic acid

Km Michaelis constant S entropy S substrate LDL low-density lipoprotein Lp(a) lipoprotein(a); T temperature TG triglycerides mRNA messenger ribonucleic acid THF tetrahydrofolate MTHF methylene tetrahydrofolate TMP thymidine monophosphate MTHFR methylenetetrahydrofolate reductase TMPT thiopurine methyltransferase TPN total parenteral nutrition NAD+ nicotinamide adenine dinucleotide TPP thiamine pyrophosphate (oxidised form) TRH thyrotrophin-releasing hormone NADH nicotinamide adenine dinucleotide tRNA transfer ribonucleic acid (reduced form) TSH thyroid-stimulating hormone NADP+ nicotinamide adenine dinucleotide phosphate (oxidised form) UDP uridine diphosphate NADPH nicotinamide adenine dinucleotide phosphate (reduced form) UMP uridine monophosphate UTP uridine triphosphate

P site peptidyl site P product VLDL very-low-density lipoprotein V maximum rate (velocity) of reaction Pi inorganic phosphate max

PIP2 phosphatidylinositol 4,5-bisphosphate xii

Acknowledgements

Thanks to the following medical students for their help reviewing chapters: Jessica Dunlop, Aliza Imam, Roxanne McVittie, Daniel Roberts and Joseph Suich. 93

Chapter 4 Carbohydrates

Introduction...... 93 pathway...... 107 Case 3 Collapsed and The citric acid cycle ...... 110 unresponsive...... 94 Oxidative phosphorylation...... 112 Chemical energy ...... 96 Glycogen...... 116 Glucose breakdown...... 102 Gluconeogenesis...... 119 The pentose phosphate Other fuels: fructose and galactose . 121

Starter questions

Answers to the following questions are on page 122 . 1 . Why is fructose a ‘quicker’ source of energy than glucose?

2 . What is the difference between the d- and l-forms of glucose? 3 . Why is the brain solely dependent on glucose for energy? 4 . Is muscle burn caused by a build-up of lactic acid? 5 . Why are mitochondria considered the powerhouses of cells? 6 . Why does cyanide poisoning work so quickly?

Introduction Carbohydrates are molecules containing and fat. Neither are carbohydrates essential carbon, hydrogen and oxygen. As the name for the synthesis of other molecules. How- implies, they are generally hydrates of car- ever, they are more readily converted than bon, and as in water, they contain hydrogen protein or fat into the key energy monosac- and oxygen in a ratio of 2:1. They are structur- charide: glucose. ally termed saccharides (Latin: saccharum, When digested, all carbohydrates are bro- ‘sugar’) and are divided into four chemical ken down to glucose, which is then transport- groups: mono-, di-, oligo- and polysaccha- ed in the blood to cells for energy production. rides. Carbohydrate is one of the main products Although a common source of energy, of photosynthesis in plants, predominantly carbohydrates are not an essential nutrient, glucose and is the main fuel for cellular res- because humans are able to obtain 100% of piration to produce energy, carbon dioxide their daily energy requirement from protein and water. 94 Chapter 4 Carbohydrates

Case 3 Collapsed and unresponsive

Presentation Analysis Charles Lee, aged 32 years, presents to Dr The blood test results Table 4.1( ) show: Sloane in the emergency department. Mr ■■ low serum glucose (hypoglycaemia) Lee has collapsed and is unresponsive. ■■ markedly increased g-glutamyl He has a long history of self-neglect and transferase alcohol misuse. Clinical examination identified a pale, underweight man who felt clammy to the touch. Dr Sloane smelt alcohol. Blood test results As Mr Lee was unresponsive he was un- Reference able to question him to his lifestyle and Test Result any current medications. There was no range sign of trauma and blood pressure and Glucose (mmol/L) 1.9 3.5–5.0 heart rate were stable (110/70 mmHg and g-Glutamyl transferase (U/L) 352 < 35 65 bpm respectively). Potassium (mmol/L) 2.6 3.5–5.3 Dr Sloane orders blood tests to assess Magnesium (mmol/L) 0.40 0.70–1.0 Mr Lee’s nutritional status and liver func- tion. Based upon his history the collapse Phosphate (mmol/L) 0.34 0.7–1.40 is most likely to be the result of alcohol- Table 4.1 Blood test results for a man who has dependent nutritional deficiencies and collapsed and is unresponsive liver dysfunction.

Hypoglycaemia: causes, investigation and treatment

The emergency department is contacted by the Hypoglycaemia is a life laboratory with severely abnormal biochemistry threatening condition I’ll get bloods for insulin and results on one of their patients with ve main causes C-peptide, to rule out exogenous ↑ Insulin insulin overdose or insulinoma Charles Lee has a very low glucose of 1.9 mmol/L

↓ Glucagon

He’s also unresponsive, Charles is treated immediately with we’ll review him IV dextrose. Blood samples are also immediately taken whilst he is hypoglycaemic, to His blood ethanol investigate the cause is high too ↓ Cortisol How does that cause Alcoholics are often malnourished Hypoglycaemia hypoglycaemia? and have low glycogen stores The insulin and C-peptide are appropriately low. His alcohol abuse is the most likely Alcohol cause Also, ethanol metabolism increases the NADH/NAD+ ratio in the cell, ↓ Glycogen which impairs gluconeogenesis

Medications The cause of his hypoglycaemia is discussed at ward round Case 3 Collapsed and unresponsive 95

Case 3 continued ■■ low glycogen stores ■■ impaired citric acid cycle and ■■ decreased potassium (hypokalaemia), gluconeogenesis; ethanol metabolism magnesium (hypomagnesaemia) and by alcohol dehydrogenase reduces phosphate (hypophosphataemia) NAD+ to NADH; the next step in the In this clinical context, the probable expla- metabolism of alcohol by aldehyde nations are hypoglycaemia reflecting dehydrogenase also reduces NAD+ to the inability to mount a glyconeogenetic NADH thereby increasing the NADH/ response due to a malnourished state, high NAD+ ratio. This change affects g-glutamyl transferase as a result of alco- gluconeogenesis holic liver damage, and lowered electro- This change in NADH negatively affects lytes secondary to nutritional deficiencies. enzymes in the gluconeogenesis pathway. Conversion of lactate to pyruvate is inhib- Further case ited and lactate production is favoured; To aid Dr Sloane’s differential diagnosis production of oxaloacetate from malate further laboratory tests were requested on is inhibited. However other mechanisms Mr Lee to measure insulin and C-peptide. for glucose production such as glycoge- Both were undetectable indicating a non- nolysis can still occur. Mr Lee has a poor insulin mediated hypoglycaemia. nutritional intake so glycogen stores are Mr Lee is admitted to hospital for intra- minimal. Alcohol misuse is associated venous replacement of potassium, mag- with nutritional deficiencies resulting nesium and phosphate. Glucose is also from a poor diet and inhibition of diges- given intravenously. After several hours tive enzymes by alcohol will impair nutri- he is sitting up in bed, alert and chatting ent absoprtion. These lead to electrolyte to the doctor. deficiencies requiring oral or intravenous supplementation. Mr Lee’s collapse was caused by hypo- Further analysis glycaemia secondary to alcohol inhibit- The insulin and C-peptide measurements ing gluconeogenesis. He is referred to an were indicated by Mr Lee’s hypoglycae- alcohol withdrawal programme, advised mia to confirm that excess insulin is not on eating and lifestyle changes and pre- responsible for his condition. C- (con- scribed multi-vitamins to help correct necting-) peptide is a by-product of the nutritional deficiencies. breakdown of proinsulin to active insu- lin, therefore its concentration is used to assess insulin production. g-Glutamyl transferase concentration Hypoglycaemia is common in patients is increased in 75% of patients with who consume excessive amounts of al- long-term alcohol misuse. This result cohol without adequate nutrition. In this indicates oxidative damage to the group of patients, the condition is a con- liver . sequence of: 96 Chapter 4 Carbohydrates

Chemical energy Chemical energy is energy stored in the Monosaccharides bonds of chemical compounds. This energy is released in chemical reactions. Monosaccharides are the simplest carbohy- Food is an example of stored chemical en- drates, because they cannot be hydrolysed to ergy. Digestion breaks down the molecules smaller carbohydrates. They all have the gen- in food into smaller components. Energy is eral formula (C.H2O)n and include glucose, released by the breaking of bonds during the fructose and galactose. chemical reactions in this process. The following characteristics are used to Carbohydrates are the central molecules classify monosaccharides (Figure 4.1). of metabolic energy in all forms of organ- ■■ Carbonyl group: if this is an aldehyde, the ism; they are used to both store and generate monosaccharide is an aldose energy. Their widespread use is probably ■■ Number of carbon atoms: three for partly the consequence of the development trioses, four for tetroses, five for pentoses of photosynthesis by cyanobacteria about and six for hexoses 3.6 billion years ago. Photosynthesis har- ■■ Chiral nature: the classification of d nesses the energy of sunlight to form chemi- or l is based on the orientation of the cal bonds, and glucose is one of its products. asymmetrical carbon furthest from the carbonyl group

Food energy ■■ d-sugars have a hydroxyl group on the Energy in food is released by the metabolic right pathways described in this chapter, and ■■ l-sugars have a hydroxyl group on the is measured in kilocalories or kilojoules left (easily remembered as ‘l’ for left) (Table 4.2). Carbohydrate, protein and fat As an example, under this classification differ in structure, so they are broken down d-glucose is an aldohexose. in different pathways. However, their metab- Monosaccharides, predominately glu- olites enter shared pathways, the final com- cose, are the major source of fuel for me- mon pathways that release energy. tabolism. They are broken down in the cy- The production, storage and use of energy toplasm by the citric acid cycle, then further are fundamental to cells; they also require utilised by mitochondria to produce adenos- energy to do all their work. Therefore an un- ine triphosphate (ATP). When not required, derstanding of biochemical energetics pro- vides the basis for an understanding of me- tabolism (see page 31). Structure of d-glucose O H | | | C

Energy content of foods H C OH

Component Energy (kcal/g) Energy (kJ/g) HO C H Carbohydrate 4 17 H C OH Fat 9 37 H C OH

Protein 4 17 CH2OH Fibre 2 8 Figure 4.1 Structure of d-glucose. Alcohol 7 29 Monosaccharides are classified by carbonyl group The joule is the most widely SI unit used to measure (orange), number of carbon atoms and chiral energy nature. The classification of d or l is based on the orientation of the asymmetrical carbon furthest Table 4.2 Energy content of major food groups from the carbonyl group (blue). Chemical energy 97 monosaccharides are converted to space- efficient, insoluble storage polysaccharides A kilocalorie is commonly referred to such as glycogen. as a calorie, and is the energy required to increase the temperature of 1 g of water by 1°C . A kilojoule is equivalent Disaccharides to 4 .2 kilocalories . Disaccharides are two monosaccharaides joined by a covalent bond formed by a dehy- dration reaction. The reaction results in the loss of hydrogen from one monosaccharide Energy transfer and a hydroxyl group from the other. The body transfers energy by breaking and making bonds. Glucose is the main molecule ■■ Sucrose (table sugar) is a disaccharide formed from d-glucose and d-fructose of energy exchange. However, it is ATP, and the phosphate−phosphate bonds it contains, ■■ Lactose is formed from d-galactose and d-glucose that directly supplies chemical reactions with the energy they need.

Oligosaccharides Adenosine triphosphate Oligosaccharides are polymers of mono- Adenosine triphosphate (ATP) is the body’s saccharides. They are generally O-linked to main currency of energy; it is an adenosine amino acid side chains in proteins or lipids. molecule (adenine ring and a ribose sugar) O-linked glycosylation is when glycans (sug- attached to three phosphate groups (Fig- ars) are attached to the hydroxyl oxygen of the ure 4.2). Energy is released by hydrolysis of the amino acids serine, threonine, tyrosine or 3rd phosphate group, whose removal produces hydroxylysine and hydroxyproline. The gly- adenosine diphosphate (ADP). ADP absorbs cans can also be O-linked to oxygens on lipids. energy during cellular processes and thus Oligosaccharides differ from polysaccha- regains a phosphate group, regenerating ATP. rides in the number of linked monosaccharide units. Oligosaccharides contain two to ten, whereas polysaccharides contain hundreds. The total amount of ATP in the human body is about 0.1 mol. The energy used daily by an average-sized adult requires Sweetness is caused by the activation hydrolysis of 200–300 mol of ATP . To of taste receptors on the tongue achieve this, each molecule of ATP is by hydroxyl groups with certain recycled 2000–3000 times during the day . orientations. Through evolution, humans developed their sensitivity and attraction to sweetness because of its Phosphates are high-energy molecules. They value as a readily metabolised source of abundant energy . release high levels of energy when individual phosphate groups are removed.

ATP structure Figure 4.2 Structure of adenosine triphosphate.

High-energy NH2 anhydride linkages N N O O O Ester linkage || || || O ¯O — P — O — P — O — P — O — CH2 N N | | | – – – O O O Adenine H H γ β α H H

Three phosphate OH OH groups Ribose 98 Chapter 4 Carbohydrates

Energy for reactions Redox coenzymes The dephosphorylation reaction of ATP to In metabolism, specific enzymes catalyse ADP is frequently coupled to another reac- oxidation. Cofactors of these enzymes, tion to exploit the release of energy. Release (coenzymes), usually act as carriers for the of phosphate is exothermic (gives off heat) products of reactions. and can be joined to an endothermic reac- In metabolism, three key coenzymes are tion (one that requires energy or heat). The responsible for the transduction of energy: phosphate group is transferred to another ■■ nicotinamide adenine dinucleotide compound (phosphorylation) to produce (NAD+) ADP, phosphate and energy. ■■ flavin adenine dinucleotide (FAD) The phosphate bond in ATP contains ■■ flavin mononucleotide (FMN) sufficient energy to supply most reactions. When more energy is needed, ATP is able to During energy metabolism, i.e. the series of release two phosphate groups. This produces reactions that produce or use ATP, electrons adenosine monophosphate (AMP) and pyro- are transferred from carbohydrates and fats phosphate, which has two phosphate groups. to these coenzymes, thereby reducing them.

The reduced products are NADH, FADH2 and Other energy currencies FMNH2, respectively. + Although ATP is the main energy carrier, The hydrogen gained by NAD from other nucleotide phosphates are also used NADH can be transferred to FAD. The re- as energy currencies. The citric acid cycle duced FADH2 enables NADH to return to its + rephosphorylates only ADP. However, it can oxidised form (NAD ). Electrons are moved transfer phosphate from ATP to a nucleo- through a sequence of reactions using the co- tide diphosphate, commonly guanosine enzymes to enable the transfer of hydrogen diphosphate (GDP) (to form guanosine tri- to oxygen, forming water. phosphate, GTP). GTP can be used to supply energy for protein synthesis and gluconeo- genesis. It is also essential in G-protein− Electron transport linked signal transduction (see page 19). system and oxidative phosphorylation Electron transport and This chain of events is the electron trans- oxidative phosphorylation port system. The system also drives the transport of protons from the mitochon- In mitochondria, the main site of ATP pro- drial matrix to the intermembrane space of duction, coenzymes transfer the energy from mitochondria. This proton transport gen- products of metabolism to ATP. They are erates potential energy in the form of a pH known as redox coenzymes, because they gradient and an electrical potential across couple reduction and oxidation reactions. In the membrane. Protons are able to flow simple terms, they are vital to the transfer of back across the membrane through ATP electrons and protons, and during this pro- synthase. cess produce a large amount of free energy The ATP synthase uses the energy to gen- used to form ATP from ADP. erate ATP from ADP in a phosphorylation reaction; this process is known as oxidative Remember that an oxidation reaction phosphorylation. Oxidative phosphorylation involves loss of hydrogen or electrons, is the metabolic pathway by which the mi- and reduction is gain of hydrogen or electrons. A simple mnemonic is OIL RIG tochondria, the enzymes and the energy re- (oxidation is loss, reduction is gain) . leased during the electron transport system reform ATP (see page 113). Chemical energy 99

Pathways of energy ■■ Glycogen breakdown: glycogenolysis metabolism (when glucose levels are low) The coordination of these pathways is called Carbohydrate metabolic pathways are the cen- glucose homeostasis. tral pathways of energy metabolism. They are based on the synthesis, storage and production of glucose (Figure 4.3). The energy generated is Tubes used to collect blood samples used to drive other biochemical reactions. for the measurement of glucose Glucose circulates in the bloodstream, concentration (a very common test) and its availability determines which of the contain an inhibitor of glycolysis. This inhibitor, usually fluoride, prevents carbohydrate pathways are active. Glucose still-active red blood cells from breaking availability is determined by the following down the glucose present in the blood . processes. ■■ Glycolysis: the breakdown of glucose ■■ The pentose phosphate pathway: the Glucose homeostasis conversion of glucose to the sugars used to synthesise nucleotides and nucleic acids; Cells in the Islets of Langerhans monitor glu- this generates no ATP but a large amount cose levels. Blood glucose concentration is of the reduced form of nicotinamide kept within a normal range of 3.5–6.0 mmol/L adenine dinucleotide phosphate (NADPH) mainly by the action of two pancreatic hor- ■■ Gluconeogenesis: the synthesis of glucose mones: glucagon and insulin. from non-carbohydrates when levels are ■■ Glucagon is released by pancreatic alpha low cells when glucose levels are low. It ■■ The electron transport system and stimulates glycogenolysis in liver cells, oxidative phosphorylation: the use of and gluconeogenesis in liver and kidney glucose to produce ATP cells, when glycogen is depleted ■■ Glycogen production: glycogenesis (when ■■ Insulin is released from pancreatic beta glucose levels are high) cells when glucose levels are high, and

Figure 4.3 Overview of Carbohydrate metabolism carbohydrate metabolism.

Glycogen

Glycogenesis Glycogenolysis Pentose phosphate pathway Pentose and Glucose other sugars

Gluconeogenesis Glycolysis

Certain Pyruvate amino acids

Anaerobic Aerobic

Acetyl Lactate Fatty acids coenzyme A

Citric acid cycle Electron transport system

CO2 H2OATP 100 Chapter 4 Carbohydrates

acts on all cells to increase glucose uptake Diabetes by facilitated diffusion through glucose Diabetes is a group of metabolic diseases pre- transporter type 4 (GLUT4) membrane dominantly in which either the production of transporters; insulin also acts on liver insulin is deficient (type 1 diabetes) or cells cells to increase glycogenogenesis have increased resistance to it (type 2 diabe- Other hormones also influence blood glucose tes) (Table 4.4). Both these types of diabetes levels (Table 4.3). increase blood glucose, which will result in Disorders of glucose homeostasis will the following: result in either hypoglycaemia (low blood ■■ Glycosuria: glucose in urine, as the renal glucose) or hyperglycaemia (high blood glu- threshold for glucose reabsorption is cose). Signs and symptoms of both are shown exceeded in Figure 4.4.

Hormones that affect blood glucose

Hormone Tissue of origin Metabolic effect(s) Effect on blood glucose Insulin* Pancreas (beta Stimulates glycogenesis Decrease cells) Enhances glucose uptake by cells Antagonistic to gluconeogenesis Enhances synthesis of fatty acids and proteins Glucagon* Pancreas (alpha Stimulates glycogenolysis Increase cells) Enhances synthesis of glucose from non- carbohydrate precursors (amino acids and fatty acids) Cortisol Adrenal cortex Stimulates gluconeogenesis Increase Facilitates activation of glycogen phosphorylase (essential for the effects of adrenaline on glycogenolysis) Antagonises insulin Adrenaline Adrenal medulla ‘Fight or flight’ response Increase (epinephrine) Enhances glycogenolysis Increases release of fatty acids from adipose tissue Growth hormone Anterior pituitary Antagonises insulin Increase Decreases rate at which cells use carbohydrate for energy Somatostatin (growth Pancreas (delta Inhibits secretion of insulin and glucagon Decrease hormone−inhibiting cells) hormone) Thyroxine Thyroid gland Enhances glycogenolysis Increase Stimulates the use of carbohydrates as an energy source *Main hormones in glucose homeostasis.

Table 4.3 Hormones that affect blood glucose concentration Chemical energy 101

Key features of hypo- and hyperglycaemia

Hyperglycaemia Hypoglycaemia

Sleepy Blurry vision Anxious or tired Dizzy Sweating

Headache Blurry vision

Polydipsia Pale Tachycardia Dry skin Infections and poor wound healing

Hungry Tremor Weak Polyuria Hungry

Figure 4.4 Key features of hypoglycaemia and hyperglycaemia.

■■ Polyuria: excess water excretion as it ‘follows’ the hypertonic urine with excess Diabetes causes long-term complications glucose from glucose-induced vascular disease. ■■ Dehydration: decreased blood volume as This condition results from high levels of water is lost from the blood, followed by glucose damaging structural proteins, disrupting fluid balance and generating water leaving the cells to maintain fluid increased cell damage from free radicals balance (reactive atoms or molecules with an ■■ Polydipsia: increased thirst unpaired electron that are a by-product of an overwhelmed electron transport system) . Complications of chronic glucose damage include poor wound healing, retinopathy, nephropathy and neuropathy, as well as cardiovascular and cerebrovascular disease . 102 Chapter 4 Carbohydrates

Classification of diabetes

Type Description Information on groups affected Treatment Type 1 diabetes Autoimmune disease that Onset usually in childhood Insulin destroys pancreatic beta cells (juvenile diabetes) Absence or deficiency of insulin production Type 2 diabetes Insulin resistance: insulin is Previously called adult onset Dietary modification and produced but not used as diabetes, but with the increasing exercise effectively prevalence of obesity, an Oral medications increasing number of young to improve insulin adults and children are being sensitivity diagnosed Insulin Gestational diabetes Increased insulin resistance as Affects 3–5% of all pregnancies Diet and exercise a result of placental hormone and develops in 2nd and 3rd Rarely insulin production trimester Insulin requirement doubled to tripled by growth of fetus Latent autoimmune Classified as type 2 but lacks Adulthood Insulin requirement may diabetes of adulthood the classic symptoms rapidly develop (occasionally referred to as More typical physique of type 1 type 1.5 diabetes) but does not require insulin in early stages Maturity onset diabetes of Similar to type 2 diabetes, with Adults aged < 25 years Depends on type, but the young strong genetic risk most common type Not linked to obesity (caused by HNF1A gene; about 70% of More than 10 different genetic cases) treated with mutations for this condition sulfonylurea pills have been identified to increase insulin production Type 3 diabetes A form of Alzheimer’s disease Late adulthood As for types 1 and 2 that results from insulin resistance in the brain Patients with type 2 diabetes have increased risk of developing Alzheimer’s disease Steroid-induced diabetes Treatment with corticosteroids Long-term steroid treatment As for type 2 potentially will increase insulin resistance, particularly in people at high risk of developing type 2 diabetes

Table 4.4 Classification of diabetes

Glucose breakdown The breakdown of glucose (glycolysis) is of cellular respiration (the reactions in a cell essential, because glucose is the main source that produce energy from nutrients) and of energy for cells. Glycolysis is the first step is also a central metabolic pathway, with Glucose breakdown 103

intermediate metabolites that link to other acid to be excreted from the cell and to pathways including those of fat and protein regenerate the NAD+ needed for glycolysis metabolism. Glycolysis has many steps but yields only two Glycolysis occurs in the cytosol of the moles of ATP per mole of glucose. Most ATP is cells of all organisms (with rare exceptions) generated in the mitochondria from the elec- to convert one molecule of glucose to two tron transport system. pyruvate molecules, two ATP molecules, hy- drogen ions, water and heat: The glycolytic pathway occurs in d-Glucose + 2 NAD+ + 2 Pi + 2 ADP every cell of the body, because all cells catabolise glucose for energy. ↓ Other monosaccharides can also be + 2Pyruvate + 2 NADH + 2 ATP + 2 H + 2 H2O metabolised by the pathway, including + heat fructose, galactose and mannose .

The fate of pyruvate depends on whether oxy- gen is present. ■■ In the presence of oxygen, pyruvate Glycolytic pathway enters aerobic respiration and is Nine reactions convert 6-carbon glucose to converted to acetyl coenzyme A, which two 3-carbon pyruvate molecules (Table 4.5). then enters the citric acid cycle in The energy released by breaking the carbon− mitochondria carbon bond is used to produce two mole- ■■ In the absence of oxygen, anaerobic cules of ATP (from ADP) and two molecules respiration converts pyruvate to lactic of NADH (from NAD+).

Glycolysis in nine steps

Generates (+) Step Reaction Product(s) Enzyme or uses (–) ATP† 1 Phosphorylation Glucose 6-phosphate Hexokinase – 2 Conversion Fructose 6-phosphate Phosphoglucose isomerase 3 Phosphorylation Fructose 1,6-bisphosphate Phosphofructokinase − 4 Cleavage Dihydroxyacetone phosphate + Glyceraldehyde 3-phosphate (1st product) Conversion Glyceraldehyde 3-phosphate (2nd Triose phosphate isomerase product) 5* Oxidation coupled to 1,3-Bisphosphoglycerate Glyceraldehyde 3-phosphate phosphorylation dehydrogenase 6* Dephosphorylation 3-Phosphoglycerate Phosphoglycerate kinase ++ 7* Rearrangement 2-Phosphoglycerate Phosphoglycerate mutase 8* Dehydration Phosphoenolpyruvate Enolase 9* Dephosphorylation Pyruvate Pyruvate kinase ++ ATP, adenosine triphosphate. *One molecule of glucose produces two molecules of glyceraldehyde phosphate. Steps 5–9 are for two molecules of glyceraldehyde 3–phosphate. †For one molecule of glucose, two ATP are used and four are produced, giving a net gain of two ATP.

Table 4.5 Summary of glycolysis 104 Chapter 4 Carbohydrates

Investment stage phosphoglucose isomerase. This rearranges the six-membered carbon ring into a five- The first three steps of glycolysis are the membered ring, so that carbon 1 becomes ‘investment’ stage: energy is invested in the external to the ring structure. processes for the outcome at the end of the pathway. Step 3: phosphorylation Step 1: phosphorylation Carbon 1 is phosphorylated by phosphofruc- tokinase-1 to produce fructose 1,6-bispho- The first step of glycolysisFigure 4.5 ( ) is phos- sphate. This reaction requires ATP and is phorylation of the glucose ring, a reaction irreversible. As in step 1, magnesium is catalysed by hexokinase. A phosphate group required as an enzyme cofactor. from ATP is added to carbon 6 of the glucose molecule to produce glucose 6-phosphate. Step 4: splitting This step ‘traps’ glucose in the cell, because Fructose 1,6-bisphosphate is cleaved in the the negatively charged phosphate prevents middle to produce two 3-carbon molecules. it from crossing the cell’s plasma membrane This is a freely reversible reaction producing through glucose transporters. A magnesium dihydroxyacetone phosphate and glyceral- ion is required to form a complex with ATP. dehyde 3-phosphate. Only glyceraldehyde 3-phosphate, two per molecule of glucose, Step 2: rearrangement of the continues in the glycolytic pathway, with carbon ring dihydroxyacetone phosphate converted to Glucose 6-phosphate is converted into fruc- glyceraldehyde 3-phosphate by triose phos- tose-6-phosphate in a reaction catalysed by phate isomerase.

Glycolysis: steps 1–4

HO — CH2 O OH HO OH OH Glucose ATP Mg2+ O O Hexokinase || 1 || Triose O C — H phosphate CH2 — O — P — O¯ || ADP isomerase | ¯O — P — O — CH O¯ 2 H — C — OH C || O | O O || O¯ OH CH — O — P — O¯ CH — OH OH OH 2 2 | Dihydroxyacetone OH O¯ phosphate Glucose 6-Phosphate Glyceraldehyde 3-Phosphate

Phosphoglucose 2 3 4 Aldolase isomerase 2+ O Mg O O || Phosphofructokinase-1 || || O — P — O — CH – 2 O CH — OH ¯O — P — O — CH O CH — O — P — O | 2 2 2 OH | OH | O¯ O¯ O¯ OH ATP ADP OH OH OH Fructose 6-Phosphate Fructose 1, 6-Phosphate

Figure 4.5 Steps 1–4 of glycolysis: the investment stage. One mole of glucose is converted to two moles of glyceraldehyde 3-phosphate. ADP, adenosine diphosphate; ATP, adenosine triphosphate. Glucose breakdown 105

Yield stages The steps are linked, because energy released from the first exergonic oxidation reaction The next steps of the pathway Figure 4.6( ) are drives the second endergonic phosphoryla- considered the ‘yield’ stages: 4 moles of ATP tion reaction. are produced for an overall gain of 2 moles of ATP, because 2 were used in steps 1 and 3. Step 6: dephosphorylation Step 5: oxidation and Next, 1,3-bisphosphoglycerate is converted phosphorylation to 3-phosphoglycerate by phosphoglycer- ate kinase, which removes a phosphate and Glyceraldehyde 3-phosphate is converted to transfers it to ADP to produce ATP. This is the 1,3-bisphosphoglycerate by glyceraldehyde first step in the production of ATP, with two 3-phosphate dehydrogenase in two reactions moles of ATP produced for each mole of glu- (Figure 4.7). cose. Two moles were used in steps 1 and 3, 1. Oxidation of glyceraldehyde 3-phosphate so at this point in the pathway there is a net at the carbon 1 position and simultaneous balance of zero ATP. reduction of NAD+ to NADH 2. Phosphorylation: transfer of phosphate Step 7: phosphate rearrangement from 1,3-bisphosphoglycerate to Phosphoglycerate mutase moves the posi- the oxidised form of glyceraldehyde tion of a phosphate so that 3-phosphoglycer- 3-phosphate ate becomes 2-phosphoglycerate. Initially a

Glycolysis: steps 5-9

5 6 O O O O Phosphoglycerate || Glyceraldehyde || || || kinase C — H 3-Phosphate C — O — P — O¯ C — O¯ | | dehydrogenase | Mg2+ | H — C — OH O H — C — OH O ¯ O H — C — OH O | || | || | ||

CH2 — O — P — O¯ CH2 — O — P — O¯ CH2 — O — P — O¯ | + + | | NAD + Pi NADH + H ADP ATP O¯ O¯ O¯ Glyceraldehyde 1,3-Bisphosphoglycerate 3-Phosphoglycerate 3-Phosphate

Pi 7 Phosphoglycerate mutase Pi

+ H2O O O O ATP ADP + H+ || || || C — O¯ O C — O¯ O C — O¯ | || Enolase | || | H — C — O — P — O¯ H — C — O — P — O¯ C || O Pyruvate kinase || | | 8 OH O¯ CH2 2+ CH2 O¯ CH3 Mg 9 Pyruvate Phosphoenolpyruvate 2-Phosphoglycerate

Figure 4.6 Continuation of the glycolytic pathway (steps 5–9): the yield stages. Only the processing of one glyceraldehyde 3-phosphate is shown, but for every glucose molecule entering the pathway, two glyceraldehyde 3-phosphates are produced. ADP, adenosine diphosphate; ATP, adenosine triphosphate; NAD+, nicotinamide adenine dinucleotide (oxidised form); NADH, nicotinamide adenine dinucleotide (reduced form), Pi, inorganic phosphate. 106 Chapter 4 Carbohydrates

Figure 4.7 The glyceraldehyde Glyceraldehyde 3-phosphate dehydrogenase reaction 3-phosphate dehydrogenase 1 Glyceraldehyde reaction. The active site of the 3-Phosphate sulfhydryl group of the enzyme dehydrogenase forms a thiohemiacetal adduct | H O with glyceraldehyde 3-phosphate. C 2 The thiohemiacetal is oxidised | | | OH to a thioester by nicotinamide R SH S — C adenine dinucleotide (NAD+), | R 1 also bound in the active site. 3 NAD+ NAD+ H Phosphate enters the active site and displaces the thiol group through a phosphorylase reaction; this yields 1,3-bisphosphoglycerate NADH 2 and regenerates the sulfhydryl NAD+ 4 group. 4 The enzyme exchanges NADH for NAD+, thus completing O the cycle. Pi, inorganic phosphate. || R — C — O — P | | O SH S — C R 3 NADH NADH Pi

phosphate is added to carbon 2, and then the The fate of pyruvate depends on the cell group on carbon 3 is removed. type, whether mitochondria are present, and the amount of oxygen available. Step 8: dehydration The NAD+ used to oxidise glyceraldehyde The 2-phosphoglycerate is dehydrated (i.e. 3-phosphate in step 5 is essential in glycoly- loses a water molecule) to become phospho- sis, therefore the resulting NADH must be enolpyruvate in a reaction catalysed by eno- reoxidised to NAD+. This occurs in both the lase. aerobic and the anaerobic pathways of pyru- vate metabolism. Step 9: dephosphorylation The final step of glycolysis converts phospho- enolpyruvate into pyruvate with the help of Mature red blood cells contain pyruvate kinase. This enzyme transfers the no mitochondria. Therefore they phosphate group from carbon 2 to ADP, yield- rely entirely on glycolysis and the ing ATP. Because there are two molecules of subsequent anaerobic metabolism of pyruvate for their energy . phosphoenolpyruvate, two molecules of ATP are generated. End of glycolysis Anaerobic metabolism of By the end of glycolysis, for every molecule pyruvate of glucose that entered the pathway there In anaerobic conditions, such as poor perfu- is a net gain of two moles of ATP, because sion or in muscle during oxygen depletion, 4 were generated but 2 were used. The nine lactate dehydrogenase transfers the hydro- steps of glycolysis are anaerobic, i.e. oxygen gen molecule from NADH to pyruvate (Fig- is not required to produce pyruvate. ure 4.8) to produce lactate and NAD+. The The pentose phosphate pathway 107

carbon double bond in pyruvate is reduced to Production of lactate and oxidised a hydroxyl group (C–O–H) in lactate. nicotinamide adenine dinucleotide All cells have lactate dehydrogenase, be- (NAD+) cause it is an essential reaction to restore O O cellular NAD+ in the absence of oxygen. || Lactate || C — O¯ dehydrogenase C — O¯ | |

C || O HO — C — H Lactate is cleared by the lactic acid (or | | Cori) cycle. It leaves the muscle cells + + CH3 H + NADH NAD CH3 via shuttling into the bloodstream and Pyruvate Lactate is converted in the liver to glucose by gluconeogenesis . a Lactate dehydrogenase Pyruvate Lactate Aerobic metabolism of H+ + NADH NAD+ pyruvate

1,3-Bisphospho- Glyceraldehyde In cells with mitochondria and in the pres- glycerate Glyceraldehyde 3-Phosphate ence of enough oxygen, pyruvate is convert- 3-Phosphate dehydrogenase ed by the citric acid cycle (see page 110) into carbon dioxide and water. It is initially con- b Step 5 of glycolysis verted to acetyl coenzyme A, which enters the cycle to produce carbon dioxide. Figure 4.8 Production of lactate and oxidised nicotinamide adenine dinucleotide (NAD+). (a) The NADH and FADH2 produced in the Anaerobic metabolism of pyruvate to produce cycle then enter the electron transport sys- lactate. (b) Recycling of reduced nicotinamide tem to form water and energy as ATP (see adenine dinucleotide (NADH) during anaerobic page 113). The NADH is reoxidised in the glycolysis, which enables regeneration of NAD+ for mitochondria to NAD+, ready for glycolysis. use in step 5 of the glycolysis pathway. Aerobic metabolism is up to 15 times more efficient than anaerobic metabolism.

The pentose phosphate pathway The pentose phosphate pathway, also known NADPH as the hexose monophosphate shunt, occurs in the cytosol of all cells. It is an alternative The reduced form of nicotinamide adenine pathway for glucose oxidation, and although dinucleotide phosphate is an important it generates no ATP, it produces over half the coenzyme that prevents oxidative stress in body’s NADPH. cells. NADPH achieves this by donating elec- The main role of the pentose phosphate trons used to reduce glutathione, in a reac- pathway is to produce five carbon sugars tion catalysed by glutathione reductase. (pentoses) used in nucleotide and amino acid Reduced glutathione is subsequently synthesis, as well as the NADPH necessary used to reduce damaging hydrogen perox- for reductive biosynthetic reactions, includ- ide (H2O2) to water, in a reaction catalysed by ing the production of fatty acids. The pentose glutathione peroxidase. Without NADPH, re- phosphate pathway is most active in the liver, active H2O2 is converted to free radicals that adrenal cortex and mammary glands . damage many components of the cell. 108 Chapter 4 Carbohydrates

The pentose phosphate Pentose phosphate pathway: pathway and glycolysis oxidative phase

The pathway branches from the glycolytic Glucose 6-Phosphate pathway (see Figure 4.5) after step 1 after pro- NADP+ duction of glucose 6-phosphate. It is a ‘shunt’ Glucose 6-Phosphate dehydrogenase through its link to glycolysis, because when NADPH + H+ pentoses are not needed for biosynthetic reactions, the pentose phosphate intermedi- 6-Phosphogluconic ates are shunted back into the main glycolysis acid lactone pathway by conversion into fructose 6-phos- H2O phate and glyceraldehyde 3-phosphate. Lactonase H+

Synthesis of deoxyribonucleic acid 6-Phosphogluconate (DNA) and ribonucleic acid (RNA) depends on the pentose phosphate NADP+ 6-Phosphogluconate pathway. This is because the nucleotides dehydrogenase that form their backbone are based on NADPH + H the 5-carbon sugars ribose (in RNA) and deoxyribose (in DNA) . 3-Keto-6 Phosphogluconate

H+

Stages of the pentose CO2 phosphate pathway Ribulose 5-Phosphate The pentose phosphate pathway has two stages. Figure 4.9 The oxidative phase of the pentose 1. An irreversible redox (reduction−oxidation phosphate pathway. These initial reactions are reaction) stage produces NADPH and irreversible and produce two nicotinamide adenine pentose phosphates (Figure 4.9) dinucleotide phosphate (reduced form; NADPH). + 2. A reversible interconversion stage converts NADP , nicotinamide adenine dinucleotide excess pentose phosphates into glycolytic phosphate (oxidised form). pathway intermediates (Figure 4.10) Step 2: hydrolysis Next, 6-phosphogluconic acid lactone is hydro- Stage 1: redox lysed to 6-phosphogluconate by lactonase. Overall, the redox stage produces two moles of NADPH per mole of glucose 6-phosphate. Step 3: decarboxylation Oxidative decarboxylation of 6-phosphoglu- Step 1: oxidation conate, catalysed by 6-phosphogluconate Glucose 6-phosphate is converted to 6-phos- dehydrogenase, produces ribulose 5-phos- phogluconic acid lactone, and NADP+ is phate through an intermediate of 3-keto- reduced to NADPH. Glucose 6-phosphate 6-phosphogluconate. dehydrogenase oxidises the aldehyde group One mole of carbon dioxide and one mole of glucose 6-phosphate to the acid. of NADPH are produced in this reaction. The pentose phosphate pathway 109

ratio of 100. Interestingly, the ratio of Pentose phosphate pathway: non- NADH:NAD+ in the cytoplasm is the reverse, oxidative phase about 0.01 – due to the requirement of NAD+ in glycolysis. These two redox systems have Ribulose 5-Phosphate different set points and are metabolised by

Ribulose 5-Phosphate Ribulose 5-Phosphate different dehydrogenases; the pentose phos- 3-epimerase isomerate phate pathway enzymes will only use NADPH – generally using it as a reducing agent, whilst the glycolytic pathway dehydrogenases are Xylulose Ribose specific for NADH, where it is used as an 5-Phosphate 5-Phosphate oxidising agent. Transketolase Transketolase Stage 2: interconversion Sedoheptulose Glyceraldehyde 7-Phosphate 3-Phosphate Ribulose 5-phosphate is isomerised (i.e. interconverted) to ribose 5-phosphate in Transaldolase Transaldolase cells with active nucleic acid synthesis (see Figure 4.10), in which it is used to synthe- Erythrose Fructose sise ATP and other ribonucleotides. In non- 4-Phosphate 6-Phosphate dividing cells, three molecules of ribulose 5-phosphate are converted into two fructose 6-phosphate molecules and one glyceralde- Transketolase hyde 3-phosphate (Table 4.6). Glyceraldehyde Xylulose 3-Phosphate 5-Phosphate Shunt into cellular respiration Figure 4.10 The non-oxidative phase of the pentose phosphate pathway. Fructose 6-phosphate and glyceraldehyde 3-phosphate are converted to pyruvate by glycolysis. Pyruvate, as when produced in Glucose 6-phosphate dehydrogenase glycolysis, is then metabolised aerobically or deficiency is an X-linked recessive anaerobically, depending on cellular condi- disease. The disease causes cells to tions (see page 106). have reduced levels of NADPH and glutathione that protect against reactive oxygen species, rendering them prone to oxidative damage . Red blood cells Pyruvate dehydrogenase converts are particularly susceptible, and patients the pyruvate from glycolysis into present with haemolytic anaemia in acetyl coenzyme A, which can then response to infections or the use of enter the citric acid cycle. Pyruvate certain medications, such as aspirin . dehydrogenase deficiency is a congenital deficiency that results in Glucose 6-phosphate dehydrogenase, a lack of energy generated by the confers protection against malaria citric acid cycle and an accumulation infection, potentially because the of lactate . Primary biliary cirrhosis is malarial parasite is cleared more rapidly an autoimmune condition in which by the spleen due to the haemolysis . antimitochondrial antibodies against pyruvate dehydrogenase complex are detected . It manifests with jaundice, The redox stage maintains high levels of cirrhosis and xanthoma . NADPH, giving a cytoplasmic NADPH:NADP+ 110 Chapter 4 Carbohydrates

Pentose phosphate pathway: non-oxidative phase

Reaction Product(s) Enzyme Ribulose 5-phosphate Ribose 5-phosphate Ribulose 5-phosphate isomerase Ribose 5-phosphate Xylulose 5-phosphate Ribulose 5-phosphate 3-epimerase Xylulose 5-phosphate + ribose 5-phosphate Glyceraldehyde 3-phosphate + Transketolase sedoheptulose 7-phosphate Glyceraldehyde 3-phosphate + Erythrose 4-phosphate + fructose Transaldolase sedoheptulose 7-phosphate 6-phosphate Xylulose 5-phosphate + erythrose Glyceraldehyde 3-phosphate + fructose Transketolase 4-phosphate 6-phosphate

Table 4.6 Summary of reactions in the non-oxidative phase of the pentose phosphate pathway

The citric acid cycle

The citric acid cycle (also known as the tricar- ■■ Fats are converted to free fatty acids, boxylic acid cycle or Krebs cycle) is the central which are oxidised in the mitochondria to pathway that interconnects the metabolic acetyl coenzyme A pathways of carbohydrates, protein and fat. It ■■ Proteolysis of proteins produces amino enables carbon atoms from amino acids to be acids, which are metabolised to acetyl converted into glucose, but those from lipoly- coenzyme A sis are unable to be diverted into gluconeo- genesis. This means that animals cannot use fat stores to make glucose; instead, in the star- Stages of the citric acid vation state, fats are broken down to ketone cycle bodies as an alternative metabolic fuel. The citric acid cycle begins with the reaction The citric acid cycle occurs in mitochon- between acetyl coenzyme A and oxaloac- dria and consists of a repeating cycle of eight etate, a 4-carbon molecule (Figure 4.11). This reactions that generate energy from acetyl forms the 6-carbon citric acid after which the coenzyme A. cycle is named. In summary, through the cycle, two of the Acetyl coenzyme A six carbons are removed as carbon dioxide to yield the 4-carbon oxaloacetate, which starts Acetyl coenzyme A is an acetyl group the cycle again. attached to a coenzyme A molecule. The Each cycle produces three NADH, one coenzyme A molecule is a large molecule FADH2 and one GTP for every molecule of containing ADP, with two side chain groups acetyl coenzyme A. to which the acetyl groups attach. It is con- sidered a carrier of acetyl groups, more Step 1: aldol condensation importantly the carbon atoms within the Acetyl coenzyme A is joined to oxaloacetate acetyl group to the citric acid cycle. Here they to form citric acid in a reaction catalysed by are oxidised for energy production. citrate synthase. Once citric acid is formed, Acetyl coenzyme A is derived from three the coenzyme A is released from the complex. metabolic precursors. ■■ Carbohydrates undergo glycolysis Step 2: dehydration to produce pyruvate, which is The second step is removal of a water mol- decarboxylated to acetyl coenzyme A by ecule from the oxaloacetate end of the pyruvate dehydrogenase molecule; the water molecule is moved to The citric acid cycle 111

The citric acid cycle

Cis-aconitate O CoASH || Citrate CH3 — C — SCOa Acetyl Citrate H C — COOH 2 Aconitase coenzyme A synthase | HO — C — COOH H2C — COOH | |

H2C — COOH H — C — COOH | Isocitrate HO — C — COOH + O || C — COOH | NAD | Oxaloacetate H H2C — COOH H2C — COOH | Isocitrate Malate CH CO NADH 2 dehydrogenase NADH + 2 dehydrogenase |

H O || C — COOH | α-Ketoglutarate NAD+ HO — C — COOH α | -Ketoglutarate dehydrogenase + H2C — COOH NAD Malate H2C — COOH | Fumarase H — C — COOH H C — C — SCoA 2 + CO || || NADH 2 H C — COOH HOOC — C — H 2 O | Succinyl H2O H2C — COOH Fumarate coenzyme A Succinate Succinyl coenzyme dehydrogenase A synthase FADH GDP 2 Succinate FAD+ GTP

Figure 4.11 The citric acid cycle (also known as the tricarboxylic acid cycle or Krebs cycle), a sequence of + eight enzymatic reactions. CoASH, coenzyme A FAD , flavin adenine dinucleotide (oxidised form); FADH2, flavin adenine dinucleotide (reduced form); NAD+, nicotinamide adenine dinucleotide (oxidised form); NADH, nicotinamide adenine dinucleotide (reduced form); SCoA, succinyl coenzyme A. carbon 4 on the molecule to form isocitrate. form succinyl coenzyme A. This decarbox- This reaction is catalysed by aconitnase, an ylation occurs with the help of NAD+, which iron-sulphur protein, which is specific for the is converted to NADH, and a-ketoglutarate movement of water from carbon 3 to carbon 4. dehydrogenase. Step 3: oxidation and decarboxylation Step 5: substrate level The enzyme isocitrate dehydrogenase then phosphorylation catalyses oxidation of the OH group at car- In step 5, succinyl coenzyme A is attacked bon 4 to yield an intermediate from which a by a free phosphate group, which releases carbon dioxide molecule is removed to form the coenzyme A and succinate. The phos- a-ketoglutarate. This third reaction gener- phate group is transferred to GDP to pro- ates NADH from NAD+. duce a molecule of GTP. GTP is similar in structure and energy to ATP, and its synthe- Step 4: decarboxylation sis occurs similarly to that of ATP. This reac- The a-ketoglutarate loses a carbon dioxide tion is catalysed by succinyl coenzyme A molecule and is replaced by coenzyme A to synthetase. 112 Chapter 4 Carbohydrates

■■ FAD+ oxidises carbon−carbon double and Cofactors are non-protein compounds triple bonds or elements which are required for ■■ NAD+ oxidises carbon−oxygen bonds biological activity. Cofactors are ionorganic ions such as magnesium The product from the step 6 reaction is fuma- or organic cofactors including heme rate. or flavin . Organic cofactors can be coenzymes or prosthetic groups . They Step 7: hydration include the following: Fumurate undergoes addition of a water mol- ■■ NADH: involved in redox reaction ecule to form L-malate, a reaction catalysed transporting electrons between two by fumarase. reactions . Key in beta oxidation, glycolysis and citric acid cycle Step 8: oxidation ■■ NADPH: reducing agent in lipid and nucleic acid synthesis and protects In the final step of the citric acid cycle, against reactive oxygen species l-malate is then oxidised to regenerate oxa- allowing regeneration of glutathione loacetate, using NAD+ to produce NADH. ■■ FADH: primary role is transport of Overall, one citric acid cycle: electrons (energy) through electron ■■ reduces three molecules of NAD+ to transport chain . NADH ■■ + reduces one molecule of FAD to FADH2 ■■ produces one molecule of GTP Step 6: oxidation These reduced coenzymes are then sub- Succinate then undergoes removal of two jected to the next stage of metabolism: oxi- hydrogens by succinate dehydrogenase. A dative phosphorylation. Ultimately, each + molecule of FAD , a coenzyme similar to NADH produces three ATP, and each FADH + 2 NAD , is reduced to FADH2 by accepting the produces two ATP. Overall 12 ATP equiva- + + two hydrogens. FAD and NAD carry out the lents are produced per mole of acetyl coen- same oxidation and reduction reactions but zyme A. work on different classes of molecule.

Oxidative phosphorylation Mitochondria are the powerhouses of cells, These redox reactions release energy, which because they are the site of both the cit- is ultimately collected to reform ATP, the ric acid cycle (in the matrix) and oxidative base unit of chemical energy used in all aero- phosphorylation, the process by which the bic organisms. vast majority of ATP is formed. Oxidative phosphorylation is a series of redox reac- tions along proteins in the inner mitochon- Transmembrane potential drial membrane: the electron transport The energy derived from the conduction of system (or ‘chain’). Electrons are conducted electrons along the electron transport sys- through these proteins, from electron donors tem is used to drive the transport of protons to acceptors, and finally passed to oxygen. (H+) from the mitochondrial matrix to the Oxidative phosphorylation 113 intermembrane space. This movement of The electron transport protons generates a transmembrane pH gra- dient and electric potential (a store of ener- system pathway gy), which is then harvested by letting them The electron transport system consists of back down their electrochemical gradient to four large protein complexes and two small the other side. independent components called ubiquinone

(coenzyme Q10) and cytochrome c. Electrons enter the electron transport sys- Harvesting the potential tem at: energy ■■ complex I (for electrons from NADH) ■■ The ‘harvest’ of energy occurs as the protons complex II (for electrons from FADH2) pass down their gradient through the large These pathways meet at ubiquinone, which transmembrane ATP synthase enzyme. This is the start of the common electron transport enzyme has a rotary subunit that is moved system. The common pathway consists of by the protons, so that energy is collected complex III, cytochrome c and complex IV. The as in a turbine. This kinetic energy is used final electron acceptor is molecular oxygen, to form a phosphate–phosphate bond by which is reduced to water. Protons are pumped hydrolysing ADP to a third phosphate group from the matrix and into the intermembrane and forming ATP. space by complexes I, III and IV (Figure 4.12).

The electron transport system in the mitochondrial inner membrane

Intermembrane space

H+ + Glycerol 3-Phosphate H H+ H+ H+ H+ H+ Complex III Complex IV Complex I e¯ (coenzyme Q-cyt (cytochrome c (NADH-Q c-reductase) cyt c oxidase) reductase) Glycerol 3-P-Q reductase e¯ FADH2 FeS cyt b e¯ cyt a

e¯ cyt c e¯ 1 e¯ QH2 e¯ e¯ FMNH 2 FeS 2+ cyt a3-Cu ½O FeS FeS 2 e¯ + Fatty acyl H FADH FADH 2 2 coenzyme A + e¯ H+ H H+ dehydrogenase H2O H+ e¯

Fatty acyl NADH Succinate coenzyme A

Complex II (succinate Q reductase) Mitochondrial matrix

Figure 4.12 The electron transport system in the mitochondrial inner membrane. FeS, iron sulphur; FADH2, flavin adenine dinucleotide (reduced form); FMN, flavin mononucleotide; FMNH2, flavin mononucleotide (reduced form); NADH, nicotinamide adenine dinucleotide (reduced form). 114 Chapter 4 Carbohydrates

Step 2: ubiquinone The amount of chemical energy (ATP) synthesised during the electron Ubiquinone is the start of the common path- transport system per electron pair way; it accepts one or two electrons from depends on when they join the chain: complex I or II and transfers them to com- ■■ an electron pair from NADH passes plex III. Ubiquinone is a small lipid-soluble through complexes I, III and IV to compound that contains a side chain of 10 produce three moles of ATP isoprene units (hence its other name, coen- ■ zyme Q ). ■ from FADH2, only two moles of ATP 10 are synthesised, because they join at complex II and bypass complex I Step 3: complex III Complex III is an oligomer of eight peptides containing cytochrome b, an iron-sulphur

Step 1a: complex I centre and cytochrome c1; it is also known as ubiquinone–cytochrome c reductase. It oxi- Complex I is also known as NADH-Q reduc- dises ubiquinone and reduces cytochrome c. tase or NADH dehydrogenase. It is a flavo- Its transport of two electrons to cytochrome c protein; it contains a nucleic acid derivative yields sufficient energy and proton move- of riboflavin called flavin mononucleotide ment to produce one mole of ATP. (FMN). FMN acts as a dehydrogenase and can transfer one or two electrons, making it a strong oxidising agent. Step 4: cytochrome c NADH is oxidised, and the electrons Cytochrome c is a small mobile haem protein, taken from it are transferred through FMN but unlike haemoglobin, it is not involved in and iron-sulphur complexes to ubiquinone. oxygen transport. Instead, it shuttles elec- Complex I contributes enough energy to the trons from complex III to complex IV. The proton gradient to produce one mole of ATP. reduction of the iron in cytochrome c from Fe3+ to Fe2+ changes its conformation so that Step 1b: complex II electrons are transferred to cytochrome a in complex IV. Complex II contains three different flavopro- teins, each of which can be a point of entry for electrons into the electron transport path- Step 5: complex IV way (Table 4.7). Complex II does not trans- Complex IV ( cytochrome c oxidase) oxidises port protons across the membrane (Figure cytochrome c, and conducts the electrons 4.12) and therefore does not contribute to the through cytochromes a and a3 of complex IV, proton gradient. before being used to reduce oxygen to water.

Complex II flavoproteins

Flavoprotein complex Oxidation step Reduction step

+ Succinate Q reductase Oxidises succinate to fumarate Reduces FAD to FADH2 + Glycerol-3-phosphate Q reductase Oxidises cytoplasmic glycerol 3-phosphate Reduces FAD to FADH2 to dihydroxyacetone phosphate

+ Fatty acyl coenzyme A Catalyses first step in mitochondrial Reduces FAD to FADH2 oxidation of fatty acids

+ FAD , flavin adenine dinucleotide (oxidised form); FADH2, flavin adenine dinucleotide (reduced form).

Table 4.7 Three flavoproteins in complex II of the electron transport pathway Oxidative phosphorylation 115

Copper is a key component of such oxidising ATP–proton gradient enzymes, because three atoms are required for the redox-active cofactor for complex IV. Complexes I, III and IV all produce protons as Complex IV pumps protons out of the mito- they pass electrons along to the next complex chondrial matrix and into the intermem- in the electron transport system. They are brane space, enabling synthesis of another pumped out of the matrix into the intermem- mole of ATP. brane space. This movement of protons cre- ates an electrochemical potential across the inner membrane. Sugar consumption has increased from The outside of the mitochondrion be- 5 kg per person per year in the 1800s to comes more acidic and more positively about 70 kg in the last decade. Excessive charged than the matrix. This creates a flux sugar consumption underlies the of protons back into the matrix along the modern epidemics of obesity, diabetes electrochemical gradient, through the ATP and vascular disease . Different theories of sugar toxicity include: synthase complex. The membrane is imper- meable to protons except through this com- ■■ an increase in reactive oxygen species, plex. which damage cells and tissues ■■ the formation of spontaneous disruptive and permanent bonds ATP synthase with proteins and lipids, including the advanced glycated end products The ATP synthase complex is an example of implicated in vascular disease rotary catalysis. It is composed of two major complexes (Figure 4.13).

Figure 4.13 Structure of The mitochondrial ATP synthase complex the mitochondrial adenosine triphosphate (ATP) synthase H+-transporting complex. The inner membrane ATP synthase Proton channel component of the complex is F0, Intermembrane space It contains the proton channel and a stalk piece (δ) through Inner a F unit 0 which protons flow. The F1-ATP mitochondrial c membrane synthase complex consists of a b ε central ϒ subunit surrounded by alternating α and β subunits. Matrix ββγ The central ϒ unit rotates in αα F1 unit β response to the proton flux, which induces conformational changes. δ ADP, adenosine diphosphate; Pi, inorganic phosphate. Pi + 3 H + Empty ADP site H+ β α

ATP β γ α α β

ADP + Pi 116 Chapter 4 Carbohydrates

■■ An inner membrane component, which

contains the proton channel (F0) Some chemicals are toxic because ■■ they disrupt the inner mitochondrial An ATP synthase complex (F1), bound to F , with alternating a and b subunits; membrane and the electron transport 0 system. Examples are rotenone (an a central g subunit physically rotates insecticide), cyanide and carbon in response to the proton flux, like a monoxide . waterwheel or turbine, and this rotation changes the shape of the a and b subunits so that they cyclically bind ADP and release ATP

Glycogen Glycogen is a branched polysaccharide of glu- meal, as well as in skeletal muscle during rest cose that is an energy storage molecule, pre- periods. dominantly in the liver and muscle cells. Small amounts are in the kidneys and intestine. Muscle has twice the amount of Glycogen is a quickly accessible source of glycogen as that in the liver. However, glucose. It contains two glycosidic linkages: unlike liver glycogen, muscle glycogen chains of α1–4-linked glucose residues with is not readily available for other α1–6 branches about every four to six resi- tissues, because myocytes are unable dues (Figure 4.14). This structure, with many to synthesise glucose 6-phosphate . Glucose 6-phosphate is necessary to branches and glucose residues, enables ready generate glucose (for export via glucose access for the enzymes that release glucose transporter proteins) and organic from glycogen. phosphate . Without the enzymes, muscle glycogen remains for use in Glycogenesis muscle tissue only . Glycogen production (glycogenesis) occurs in the liver during and immediately after a Glycogenesis pathway The pathway has four steps with four enzymes, and occurs in the cytosol of cells Glycogen structure (Figure 4.15). Like the pentose phosphate

CH2OH pathway, glycogenesis starts after step 1 of O H α-1, glycolysis: the phosphorylation of glucose to H 6-Glycosidic OH O linkage glucose 6-phosphate by hexokinase in mus- H OH cle and glucokinase in the liver. O Step 1: isomerisation CH2OH CH2OH CH2 O O H O H Glucose 6-phosphate is isomerised to glu- H H H H O OH OOOH OH O cose 1-phosphate by phosphoglucomutase. H OH H OH H OH Step 2: conversion α-1,4- Glucose 1-phosphate reacts with uridine Glycosidic triphosphate to form uridine diphosphate linkages (UDP)-glucose and pyrophosphate. This Figure 4.14 Glycogen structure, showing α-1,4 reaction is catalysed by UDP-glucose pyro- and α-1,6-glycosidic linkages. phosphatase. Glycogen 117

Figure 4.15 Pathway Pathway of glycogenesis of glycogenesis. O Glucose is transferred || to glycogen in α1–4 HO — CH2 ¯O — P — O — CH linkages by glycogen 2 O O | synthase. When the O OH || O¯ OH OH O — P — O¯ chain exceeds eight OH OH OH | residues, a glycogen- OH Phosphoglucomutase O¯ branching enzyme Glucose 6-Phosphate Glucose 1-Phosphate transfers some of the α1–4-linked UTP α UDP-Glucose sugars to an 1–6 Pyrophosphatase Pyrophosphate branch. This enables further elongation Pi Pi of both of the α1–4 HO — CH chains in turn until 2 O O O O N they become long || || OH O enough for transfer OH by debranching O — P — O — P — O — CH2 O N OH | | enzymes. Pi, inorganic O¯ O¯ phosphate; UDP, UDP-glucose × 8 Glycogen OH OH uridine diphosphate; synthase UTP, uridine triphosphate.

Core Core

Step 3: hydrolysis the active site - the allosteric site. This results in conformational change and can enhance Pyrophosphate is rapidly hydrolysed to inor- activity (allosteric activator) or inhibit activ- ganic phosphate by pyrophosphatase. ity (allosteric inhibitor). In the fed state, glycogen synthase in the Step 4: polymerisation liver is allosterically activated by glucose The final step is the action of glycogen syn- 6-phosphate and ATP. thase, which catalyses the formation of a gly- coside bond between carbon 1 of the glucose Hormonal regulation on UDP-glucose and carbon 4 of a terminal Insulin stimulates the activity of glycogen glucose residue of glycogen, releasing the synthase, and therefore glycogenesis. Insu- UDP. This process simply adds glucose on to lin is released by pancreatic beta cells when a pre-existing glycogen fragment. blood glucose is high. When there is no glycogen polymer to start Glycogen synthase is the target enzyme this cascade, glycogenin (a protein primer) of glycogenesis regulation, because UDP- will initiate the reaction. glucose pyrophosphorylase is also involved in the synthesis of glycoproteins and other Control of glycogenesis sugars. Glycogenesis is controlled by both allosteric and hormonal regulation. Glycogenolysis Glycogen is gradually degraded between Allosteric regulation meals by glycogenolysis, releasing glucose Allosteric regulation is when an effector mol- to maintain blood glucose concentration. ecule binds to a protein at a site, other than However, total hepatic stores of glycogen 118 Chapter 4 Carbohydrates

are barely sufficient for the maintenance of blood glucose during a 12-h fast. There is Pathway of glycogenolysis then a shift from glycogenolysis to de novo synthesis of glucose (gluconeogenesis).

Glycogenolysis pathway Core The pathway has four steps with four Glycogen 7 x Glucose 1- Phosphorylase enzymes, two of which cleave different types Phosphate of glucose−glucose bonds: ■■ glycogen phosphorylase, which cleaves Core the core chain α1–4 linkages Debrancher enzyme- ■■ glycogen-debranching enzyme, which transglycosylase transfers 3–glucose sections of 4-residue branches, and then cleaves the remaining Core α1–6 branch to release free glucose Debrancher enzyme- transglycosylase Step 1: debranching 1 x glucose The branching links are α1–6 linkages, cleaved Core by glycogen-debranching enzyme, which has both transglycosylase and glucosidase activ- Glucose 6-Phosphatase ity. Transglycosylase removes the last three of four glucose residues of a branch, and trans- Glucose 6-Phosphate fers them to another branch, thus exposing the final single α1–6 glucose molecule, which

is then cleaved free by glucosidase. Glucose Step 2: phosphorylation This step is phosphorylation of the termi- nal α1–4 linked glucose residue by glycogen Circulation phosphorylase (Figure 4.16). This enzyme can cleave only the terminal glucose resi- dues, uses phosphate and releases glucose Figure 4.16 Pathway of glycogenolysis. About 1-phosphate. Most glycogen breakdown is 90% of glucose is released as glucose 1-phosphate, through this activity. and the remainder as ‘free’ glucose from the α1–6 branching residues. Step 3: isomerisation Glucose 1-phosphate is converted to glu- cose 6-phosphate by phosphoglucomutase, a reverse of the initial steps in glycogenesis. Control of glycogenolysis Glycogenolysis in the liver is activated Release into the bloodstream when glucose is in demand, either during In the liver, the glucose is released from glu- the post-absorption state or in preparation cose 6-phosphate by glucose-6-phosphatase. for increased utilisation, i.e. during stress It then exits through glucose transporter states. There are three hormonal activators type 2 (GLUT2) into the blood. of glycogenolysis (Table 4.8): Gluconeogenesis 119

Hormonal control of glycogenolysis

Hormone Source Initiator Effect on glycogenolysis Glucagon Pancreatic alpha cells Hypoglycaemia Rapid activation Adrenaline (ephinephrine) Adrenal medulla Stress or hypoglycaemia Rapid activation Insulin Pancreatic beta cells Hyperglycaemia Inactivation Cortisol Adrenal cortex Stress Chronic activation

Table 4.8 Hormonal control of glycogenolysis

1. glucagon 2. adrenaline (ephinephrine) Glycogen storage diseases result 3. cortisol from defects in glycogen synthesis or breakdown. These diseases arise as a Glycogenolysis and glycogenesis are oppos- result of a dysfunctioning enzyme in ing pathways. Therefore activation of glyco- the pathway . The most common of the genolysis is coordinated with inactivation of 12 is glucogen storage disease type I glycogenesis. (von Gierke’s disease), a lack of glucose 6-phosphatase . This condition affects 1 in 50,000 to 100,000 neonates and causes: ■■ impaired liver glycogenolysis and gluconeogenesis ■■ hypoglycaemia, liver and kidney problems, lactic acidosis and hyperlipidaemia

Gluconeogenesis Gluconeogenesis is the generation of glucose Gluconeogenesis pathway from non-carbohydrate carbon substrates. It occurs primarily in the liver. The substrates The reactions are considered the reverse of are pyruvate, lactate, glycerol and a-keto glycolysis (Table 4.9). However, three steps of acids (derived from amino acids). glycolysis are irreversible and must be over- In humans, the predominant precursors are come by distinct reactions: lactate, glycerol, alanine and glutamine, which ■■ step 1 (hexokinase) account for > 90% of gluconeogenesis. These ■■ step 3 (phosphofructokinse) sources become important when liver glycogen ■■ step 9 (pyruvate kinase) is depleted, for example from strenuous exer- Gluconeogenesis uses alternative reactions cise or prolonged fasting. The end product is ad- to make these steps. If the reactions were all equate glucose for the brain and red blood cells, reversible, gluconeogenesis would be consid- which rely on glucose as their energy source. ered highly endergonic. An endergonic reac- tion is an unfavourable reaction in terms of Drugs used to treat type 2 (insulin- energy, because the reaction would require resistant) diabetes target the more energy to proceed than it would pro- gluconeogesis pathway. For example, duce, resulting in an overall net loss of energy. metformin suppresses glucose In reverse, the steps of gluconeogesis are as production by the liver, increases insulin follows. sensitivity and enhances peripheral uptake of glucose . 120 Chapter 4 Carbohydrates

Gluconeogenesis in nine steps

Generates (+) Step Reaction Product(s) Enzyme or uses (-) ATP* 1 Carboxylation Oxaloacetate Pyruvate carboxylase – ATP 2 Phosphorylation Phosphoenolpyruvate Phosphoenolpyruvate carboxykinase – GTP 3 Hydration 2-Phosphoglycerate Enolase 4 Rearrangement 3-Phosphoglycerate Phosphoglycerate mutase 5 Phosphorylation 1,3-Bisphosphoglycerate Phosphoglycerate kinase 6 Conversion Dihydroxyacetone phosphate + Glyceraldehyde 3-phosphate glyceraldehyde 3-phosphate dehydrogenase Fructose 1,6-bisphosphate Triose phosphate isomerase 7 Conversion Fructose 6-phosphate Fructose 1,6-bisphosphatase - ATP 8 Conversion Glucose 6-phosphate Phosphoglucoisomerase 9 Dephosphorylation Glucose ATP, adenosine triphosphate; GTP, guanosine triphosphate. *Per molecule of pyruvate (four ATP and two GTP molecules overall).

Table 4.9 Summary of gluconeogenesis

Step 1: conversion glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase and aldolase (see of pyruvate to Table 4.9). phosphoenolpyruvic acid This is a two-step process in which pyruvate Step 7: fructose 6-phosphate carboxylase catalyses the carboxylation of Fructose 1,6-bisphosphatase converts fruc- pyruvate to form oxaloacetate. This reaction tose 1,6-bisphosphate to fructose 6-phos- requires ATP to proceed. phate. The reaction uses one molecule of Pyruvate carboxylase is present in mito- water and produces phosphate. This step is chondria. Oxaloacetate is subsequently de- the rate-limiting step of gluconeogenesis. carboxylated and phosphorylated to produce This is in contrast to glycoslysis, in which phosphoenolpyruvate, using GTP and the en- fructose 6-phosphate is converted to fructose zyme phosphoenolpyruvate carboxykinase. 1,6-bisphosphate by phosphofructokinase-1. There is an intermediate step in these two reactions, because oxaloacetate cannot cross the mitochondrial membrane. Oxaloacetate Step 8: glucose 6-phosphate is converted into malate (in a reduction reac- Phosphoglucoisomerase converts fructose tion using NADH) by malate dehydrogenase, 6-phosphate into glucose 6-phosphate, which which can pass through the membrane into is then used in other metabolic pathways or the cytoplasm, where it is converted back continue through gluconeogenesis. to oxaloacetate (through oxidation, using NAD+), again by malate dehydrogenase. Step 9: glucose The final stage of the pathway is dephos- Steps 2–7: glycolysis reversal phorylation of glucose 6-phosphate into free The next six steps are the exact reversal of glucose and an inorganic phosphate. Glucose glycolysis, catalysed by enolase, phospho- 6-phosphate cannot be transported out of the glycerate mutase, phosphoglycerate kinase, cell until this dephosphorylation occurs. Other fuels: fructose and galactose 121

This step is not a reversal of the start of gly- By having these three regulatory steps, the colysis, in which glucose and ATP are convert- process of gluconeogenesis goes from an ed into glucose 6-phosphate by hexokinase. energonic pathway to an exergonic pathway that can occur spontaneously: Control of gluconeogenesis 2 pyruvates + 4 ATP + 2 GTP + 2 NADH + 4 H2O The three steps in the gluconeogenesis path- ↓ way (see page 119) are the regulatory stages glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ + 2 H+ in the control of gluconeogenesis. In glycoly- sis, the enzyme pyruvate kinase is replaced These key regulation steps ensure that gly- by phosphoenolpyruvate carboxykinase, colysis and gluconeogenesis are coordinat- phosphofructokinase by fructose 1,6-bispho- ed so that only one pathway is active when sphatase, and hexokinase by glucose 6-phos- required. If both directions were active, then phatase. These three key steps are vital in the overall net result would be hydrolysis of energy conservation, because: two ATP molecules and two GTP molecules ■■ when excess energy is available, per cycle. gluconeogenesis is inhibited ■■ when energy is required, the pathway will be activated

Other fuels: fructose and galactose Fructose and galactose are sugars that pro- ride of common table sugar. vide a source of glucose, independently of gly- Unlike glucose, insulin is not needed for cogenolysis and gluconeogenesis. fructose to enter cells. Instead, it enters cells by facilitated diffusion (glucose transporter 5 Fructose GLUT5). Fructose (Figure 4.17) is in honey, fruit and root vegetables, and is absorbed directly into Glycolytic pathway the bloodstream after digestion. It is one of Fructose enters the glycolytic pathway (see the three main dietary monosaccharides, page 104) through two routes. along with glucose and galactose. Combined ■■ Fructose is converted to fructose with glucose, it forms sucrose, the disaccha- 1-phosphate by fructokinase in the liver; fructose 1-phosphate is then split Figure 4.17 The structure into dihydroxyacetone phosphate and Structure of of d-fructose. glyceraldehyde by fructose 1-phosphate d-fructose aldolase, and both molecules are CH OH 2 converted into glyceraldehyde 3-phosphate C O ■■ Fructose in converted to fructose HO C H 6-phosphate by hexokinase in muscle and adipose tissue H C OH Fructose is metabolised, predominantly in H C OH the liver, by the first of these pathways. The glyceraldehyde 3-phosphate is then used to CH OH 2 replenish liver glycogen stores or for triglycer- ide synthesis. 122 Chapter 4 Carbohydrates

Figure 4.18 The structure Structure of Fructose is converted to glycogen of d-galactose. only in an energy-depleted state, as d-galactose a ready store of glucose in the liver. It O H is usually converted into liver fat . This C might explain why studies in the 1980s of fructose-based sweeteners showed H C OH that a high-fructose diet in patients with type 2 diabetes induces high HO C H levels of blood triglycerides . Increased triglyceride levels are associated with an HO C H increased risk of atheroma formation, heart disease and stroke . H C OH

CH2OH Galactose Galactose (Figure 4.18) is a monosaccha- 1. Phosphorylation by galactokinase to form ride. However, it is predominantly present as galactose-1-phosphate a disaccharide in milk (lactose) and a part of 2. Conversion of galactose 1-phosphate to polysaccharide gums in plants. glucose 1-phosphate through UDP-glucose When digested, galactose enters the circula- 3. Conversion of glucose 1-phosphate tion by cotransport with sodium, using the same to glucose 6-phosphate by transporter as glucose, i.e. glucose transporter-1. phosphoglucomutase The metabolic pathway of galactose has 4. Glucose 6-phosphate enters the glycolytic four steps. pathway (see page 104)

Answers to starter questions

1. Fructose enters glycolysis at the level of the triose phosphate intermediates. This is after the steps controlled by the regulatory enzymes hexokinase and phosphofructokinase-1. As these are the two rate limiting steps, fructose will provide a rapid source of energy in both aerobic and anaerobic cells.

2. The d and l forms of glucose are optical isomers – molecules with the same molecular formula but different chemical structures. d-glucose is the dietary form of glucose and is used as the ubiquitous fuel in metabolism. l-glucose does not occur naturally but can be synthesised. It is not used as a source of energy as hexokinase cannot phosphorylate glucose in the l form. 3. The brain almost exclusively uses glucose as its energy substrate, for example, consuming almost 75% of the liver’s production. Other energy substrates such as free fatty acids are unable to cross the blood-brain barrier. The brain has large ATP energy requirements, which it needs to power the ion pumps that maintain membrane potentials so that neurons remain in their excitable state. ATP is also needed for neurotransmitter synthesis, release and uptake and intracellular transport. 4. Anaerobic respiration producing lactic acid is likely one mechanism causing muscle burn during intense exercise. As oxygen levels become inadequate and cells are unable use mitochondria to produce enough ATP, pyruvate is instead converted to lactate in order to generate NAD+ needed for glycolysis. Lactate is a weak Answers to starter questions 123

Answers continued

acid. However, increased metabolism of ATP also results in increased proton (i.e. acid) production and the increased carbon dioxide produced in cellular respiration contributes to a marginally lower pH. The nerves near the muscles sense the resulting acidic environment as a burning sensation. 5. Mitochondria are unique and essential organelles that produce most of the cell’s energy. They do this by breaking down glucose and utilizing a high-energy electron passing along a system of 5 protein complexes in the inner membrane to generate an electrochemical gradient of protons across the membrane. As the protons finally pass down their gradient, through the ATP synthase enzyme, the electrochemical energy is converted to kinetic energy and then to chemical energy as ADP is phosphorylated to ATP. This means one molecule of glucose produces up to 38 molecules of ATP. 6. Cyanide compounds are poisonous because they bind to the iron atom in the enzyme cytochrome c oxidase, a component of the fourth complex of the electron transport system in the mitochondria. Binding of cyanide blocks the electron transport and prevents sufficient ATP production. Highly metabolic tissues like neurons and heart muscle are particularly sensitive to this lack of aerobic respiration. Death will occur within seconds.