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BiochemistryThird Edition

iiSection K – Lipid metabolismBIOS INSTANT NOTESSeries Editor: B.D. Hames, School of Biochemistry and Microbiology,University of Leeds, Leeds, UKBiologyAnimal Biology, Second EditionBiochemistry, Third EditionBioinformaticsChemistry for Biologists, Second EditionDevelopmental BiologyEcology, Second EditionGenetics, Second EditionImmunology, Second EditionMathematics & Statistics for Life ScientistsMedical MicrobiologyMicrobiology, Second EditionMolecular Biology, Third EditionNeuroscience, Second EditionPlant Biology, Second EditionSport & Exercise BiomechanicsSport & Exercise PhysiologyChemistryConsulting Editor: Howard StanburyAnalytical ChemistryInorganic Chemistry, Second EditionMedicinal ChemistryOrganic Chemistry, Second EditionPhysical ChemistryPsychologySub-series Editor: Hugh Wagner, Department of Psychology, University ofCentral Lancashire, Preston, UKCognitive PsychologyPhysiological PsychologyPsychologySport & Exercise Psychology

BiochemistryThird EditionDavid Hames & Nigel HooperSchool of Biochemistry and Microbiology,University of Leeds, Leeds, UK

Published by:Taylor & Francis GroupIn US: 270 Madison AvenueNew York, NY 10016In UK: 4 Park Square, Milton ParkAbingdon, OX14 4RN 2005 by Taylor & Francis GroupFirst published 1997; Third edition published 2005ISBN: 0-4153-6778-6 (Print Edition)This book contains information obtained from authentic and highly regarded sources. Reprintedmaterial is quoted with permission, and sources are indicated. A wide variety of references arelisted. Reasonable efforts have been made to publish reliable data and information, but theauthor and the publisher cannot assume responsibility for the validity of all materials or for theconsequences of their use.All rights reserved. No part of this book may be reprinted, reproduced, transmitted, or utilizedin any form by any electronic, mechanical, or other means, now known or hereafter invented,including photocopying, microfilming, and recording, or in any information storage or retrievalsystem, without written permission from the publishers.A catalog record for this book is available from the British Library.Library of Congress Cataloging-in-Publication DataHames, B. D.Biochemistry / David Hames and Nigel Hooper. – – 3rd ed.p. ; cm. – – (BIOS Instant notes)Previously published in 2000 as: Instant notes.Includes bibliographical references.ISBN 0–415–36778–6 (alk. paper)1. Biochemistry – – Outlines, syllabi, etc.[DNLM: 1. Biochemistry –– Outlines. Qu 18.2. H215b 2005] I. Hooper, N. M. II. Hames,B. D. Instant Notes. III. Title. IV. Series.QP518.3.H355 2005612'. 015 – – dc222005020354Cover image: The structure of the E.coli met-repressor/DNA-operator complexdetermined by X-ray crystallography (W.S. Somers and S.E.V. Phillips.Nature 359, 387–393, 1992). Image courtesy of Dr A. Berry, Astbury Centre forStructural Molecular Biology, University of Leeds.Editor:Editorial Assistant:Production Editor:Elizabeth OwenChris DixonKarin HendersonThis edition published in the Taylor & Francis e-Library, 2006.“To purchase your own copy of this or any of Taylor & Francis or Routledge’scollection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.”Taylor & Francis Groupis the Academic Division of T&F Informa plc.Visit our web site at http://www.garlandscience.com

C ONTENTSAbbreviationsPrefaceviiixSection A – Cell structure and imagingA1 Prokaryote cell structureA2 Eukaryote cell structureA3 Cytoskeleton and molecular motorsA4 BioimagingA5 Cellular fractionation11491824Section B – Amino acids and proteinsB1Amino acidsB2Acids and basesB3Protein structureB4Myoglobin and hemoglobinB5CollagenB6Protein purificationB7Electrophoresis of proteinsB8Protein sequencing and peptide synthesis292933374856626975Section C – EnzymesC1 Introduction to enzymesC2 ThermodynamicsC3 Enzyme kineticsC4 Enzyme inhibitionC5 Regulation of enzyme activity83839196102105Section D – AntibodiesD1 The immune systemD2 Antibodies: an overviewD3 Antibody synthesisD4 Antibodies as tools113113117122127Section E – Biomembranes and cell signalingE1Membrane lipidsE2Membrane proteins and carbohydrateE3Transport of small moleculesE4Transport of macromoleculesE5Signal transductionE6Nerve function131131138145151156167Section F – DNA structure and replicationF1DNA structureF2Genes and chromosomesF3DNA replication in bacteriaF4DNA replication in eukaryotes173173178183188

viContentsSection G – RNA synthesis and processingG1 RNA structureG2 Transcription in prokaryotesG3 OperonsG4 Transcription in eukaryotes: an overviewG5 Transcription of protein-coding genes in eukaryotesG6 Regulation of transcription by RNA Pol IIG7 Processing of eukaryotic pre-mRNAG8 Ribosomal RNAG9 Transfer RNA193193195199206208212220228235Section H – Protein synthesisH1 The genetic codeH2 Translation in prokaryotesH3 Translation in eukaryotesH4 Protein targetingH5 Protein glycosylation241241245254257265Section I – Recombinant DNA technologyI1The DNA revolutionI2Restriction enzymesI3Nucleic acid hybridizationI4DNA cloningI5DNA sequencingI6Polymerase chain reaction269269271276281286289Section J – Carbohydrate metabolismJ1Monosaccharides and disaccharidesJ2Polysaccharides and tose phosphate pathwayJ6Glycogen metabolismJ7Control of glycogen metabolism293293300304315323327330Section K – Lipid metabolismK1 Structures and roles of fatty acidsK2 Fatty acid breakdownK3 Fatty acid synthesisK4 TriacylglycerolsK5 CholesterolK6 Lipoproteins335335339346352357363Section L – Respiration and energyL1Citric acid cycleL2Electron transport and oxidative phosphorylationL3Photosynthesis367367372384Section M – Nitrogen metabolismM1 Nitrogen fixation and assimilationM2 Amino acid metabolismM3 The urea cycleM4 Hemes and chlorophylls395395399407413Further Reading419Index425

A BBREVIATIONSAACATadenineacyl-CoA cholesterolacyltransferaseACPacyl carrier proteinADPadenosine diphosphateAIDSacquired immune deficiencysyndromeAlaalanineALAaminolaevulinic acidAMPadenosine monophosphateArgarginineAsnasparagineAspaspartic acidATCase aspartate transcarbamoylaseATPadenosine 5′-triphosphateATPase adenosine triphosphatasebpbase pairsCcytosinecAMP3′, 5′ cyclic AMPCAPcatabolite activator proteincDNAcomplementary DNACDPcytidine diphosphatecGMPcyclic GMPCMcarboxymethylCMPcytidine monophosphateCNBrcyanogen bromideCoAcoenzyme ACoQcoenzyme Q (ubiquinone)CoQH2 reduced coenzyme Q (ubiquinol)CRPcAMP receptor proteinCTLcytotoxic T lymphocyteCTPcytosine triphosphateCyscysteineΔE0′change in redox potential understandard conditionsΔGGibbs free energyΔG‡Gibbs free energy of activationΔG0′Gibbs free energy under enosine 5′-triphosphatedCTPdeoxycytidine 5′-triphosphateddNTP dideoxynucleoside e DNAdeoxyribonucleic dine 5′-triphosphateredox potentialEnzyme Commissionelongation factoreukaryotic initiation factorenzyme-linked immunosorbentassayendoplasmic reticulumexternal transcribed spacerfructose 2,6-bisphosphatefast atom bombardment massspectrometryfluorescence-activated cellsorterflavin adenine dinucleotide(oxidized)flavin adenine dinucleotide(reduced)fructose bisphosphataseN-formylmethionineflavin mononucleotide (reduced)flavin mononucleotide (oxidized)fluorescence resonance energytransferN-acetylgalactosamineguanosine diphosphategreen fluorescent proteinN-acetylglucosamineglutamineglutamic acidglycineguanosine monophosphateglycosyl phosphatidylinositolG protein-coupled receptorsguanosine 5′-triphosphatehemoglobinadult hemoglobinfetal hemoglobinsickle cell hemoglobinhigh density lipoproteinhistidinehuman immunodeficiency virus3-hydroxy-3-methylglutarylheavy meromyosinheterogeneous nuclear RNA

AbbreviationshnRNPheterogeneous nuclearribonucleoproteinHPLChigh-performance liquidchromatographyhspheat shock ermediate density lipoproteinIFinitiation factorIgimmunoglobulinIgGimmunoglobulin GIleisoleucineIP3inositol 1,4,5-trisphosphateIPTGisopropyl- -DthiogalactopyranosideIRESinternal ribosome entry sitesITSinternal transcribed spacerKequilibrium constantKmMichaelis constantLCATlecithin–cholesterol acyltransferaseLDHlactate dehydrogenaseLDLlow density lipoproteinLeuleucineLMMlight meromyosinLyslysineMetmethionineMSmass spectrometrymVmillivoltmRNA messenger RNANAD nicotinamide adenine dinucleotide(oxidized)NADH nicotinamide adenine dinucleotide(reduced)NADP nicotinamide adenine dinucleotidephosphate (oxidized)NADPH nicotinamide adenine dinucleotidephosphate (reduced)NAMN-acetylmuramic acidNHPnonhistone proteinNMRnuclear magnetic resonanceORFopen reading framePAGEpolyacrylamide gel electrophoresisPCplastocyaninPCRpolymerase chain sePhephenylalaninePiinorganic phosphatepIisoelectric ciation constantprotein kinase Ainorganic pyrophosphateprolineplastoquinonephotosystem Iphotosystem IIphenylthiohydantoinubiquinone (coenzyme Q)ubiquinol (CoQH2)rough endoplasmic reticulumrelease factorrestriction fragment lengthpolymorphismRNAribonucleic acidRNaseribonucleaserRNAribosomal RNArubisco ribulose bisphosphatecarboxylaseSDSsodium dodecyl sulfateSerserineSERsmooth endoplasmic reticulumsnoRNA small nucleolar RNAsnoRNP small nucleolar ribonucleoproteinsnRNA small nuclear RNAsnRNP small nuclear ribonucleoproteinSRPsignal recognition particleSSBsingle-stranded DNA-binding(protein)TBPTATA box-binding proteinTFIItranscription factor for RNApolymerase IITFIIIA transcription factor IIIAThrthreonineTmmelting er RNATrptryptophanTyrtyrosineUDPuridine diphosphateUMPuridine monophosphateUREupstream regulatory elementUTPuridine 5′-triphosphateUVultravioletValvalineV0initial rate of reactionVLDLvery low density lipoproteinVmaxmaximum rate of reaction

P REFACEIt was perhaps a mark of how successful the second edition of Instant Notes in Biochemistry was that werecall seeing a final year student avidly reading it even as he waited to have his viva with the ExternalExaminer. Although we would strongly recommend to any student not to leave revision to such a verylate stage, this experience alone proved the value of a concise book that focused on essential biochemical information in an easily accessible format!Let us be clear. This is not a book to replace the superb all-embracing and highly detailedBiochemistry textbooks that take the reader to the cutting edge of this science. Rather, its goal is toallow the reader to cut to the heart of the matter, to see what the core information is and readily toassimilate it. For mainstream Biochemistry students, it may be seen as complementary to the largedetailed textbooks, whereas for students taking Biochemistry as an optional or elective module, itshould be welcome as a fast way to become acquainted with the main facts and concepts.This book is aimed at supporting students primarily in the first and second years of their degree,although, as we recount above, it can also serve as a welcome friend when faced with certain adversesituations even in the final year! The third edition has taken on board all of the many comments andadvice that we have gratefully received from readers and academic colleagues alike, and we havecorrected a number of errors, omissions and ambiguities. No doubt we have still missed a few; do letus know of any that you spot. This revision has necessarily reflected the many new directions thatBiochemistry has taken since the last edition, whilst also preserving coverage of the core of the subject.The book now also includes expanded coverage of cell structure and imaging, proteomics, microarrays,signal transduction, etc. As with earlier editions, we have been careful to include only the informationthat we believe is essential for good student understanding of the subject – and for rapid revision whenexams appear on the horizon. Do use the book not only to get to grips with the subject but also as aready source of elusive information. We hope and believe that you will find it as useful as paststudents told us they found the earlier editions.David HamesNigel Hooper

Section A – Cell structure and imagingA1 P ROKARYOTECELLSTRUCTUREKey NotesProkaryotesProkaryotes are the most abundant organisms on earth and fall into twodistinct groups, the bacteria (or eubacteria) and the archaea (orarchaebacteria). A prokaryotic cell does not contain a membrane-boundnucleus.Cell structureEach prokaryotic cell is surrounded by a plasma membrane. The cell hasno subcellular organelles, only infoldings of the plasma membrane calledmesosomes. The deoxyribonucleic acid (DNA) is condensed within thecytosol to form the nucleoid.Bacterial cell wallsThe peptidoglycan (protein and oligosaccharide) cell wall protects theprokaryotic cell from mechanical and osmotic pressure. Some antibiotics,such as penicillin, target enzymes involved in the synthesis of the cellwall. Gram-positive bacteria have a thick cell wall surrounding theplasma membrane, whereas Gram-negative bacteria have a thinner cellwall and an outer membrane, between which is the periplasmic space.Bacterial flagellaSome prokaryotes have tail-like flagella. By rotation of their flagellabacteria can move through their surrounding media in response tochemicals (chemotaxis). Bacterial flagella are made of the protein flagellinthat forms a long filament which is attached to the flagellar motor by theflagellar hook.Related topicsProkaryotesEukaryote cell structure (A2)Cytoskeleton and molecular motors(A3)Amino acids (B1)Membrane lipids (E1)Membrane proteins andcarbohydrate (E2)Genes and chromosomes (F2)Electron transport and oxidativephosphorylation (L2)Prokaryotes are the most numerous and widespread organisms on earth, andare so classified because they have no defined membrane-bound nucleus.Prokaryotes comprise two separate but related groups: the bacteria (or eubacteria) and the archaea (or archaebacteria). These two distinct groups of prokaryotes diverged early in the history of life on Earth. The living world therefore hasthree major divisions or domains: bacteria, archaea and eukaryotes (see TopicA2). The bacteria are the commonly encountered prokaryotes in soil, water andliving in or on larger organisms, and include Escherichia coli and the Bacillusspecies, as well as the cyanobacteria (photosynthetic blue-green algae). Thearchaea mainly inhabit unusual environments such as salt brines, hot acidsprings, bogs and the ocean depths, and include the sulfur bacteria and themethanogens, although some are found in less hostile environments.

2Section A – Cell structure and imagingCell structureProkaryotes generally range in size from 0.1 to 10 μm, and have one of threebasic shapes: spherical (cocci), rod-like (bacilli) or helically coiled (spirilla). Likeall cells, a prokaryotic cell is bounded by a plasma membrane that completelyencloses the cytosol and separates the cell from the external environment. Theplasma membrane, which is about 8 nm thick, consists of a lipid bilayercontaining proteins (see Topics E1 and E2). Although prokaryotes lack themembranous subcellular organelles characteristic of eukaryotes (see Topic A2),their plasma membrane may be infolded to form mesosomes (Fig. 1). The mesosomes may be the sites of deoxyribonucleic acid (DNA) replication and otherspecialized enzymatic reactions. In photosynthetic bacteria, the mesosomescontain the proteins and pigments that trap light and generate adenosinetriphosphate (ATP). The aqueous cytosol contains the macromolecules[enzymes, messenger ribonucleic acid (mRNA), transfer RNA (tRNA) and ribosomes], organic compounds and ions needed for cellular metabolism. Alsowithin the cytosol is the prokaryotic ‘chromosome’ consisting of a single circularmolecule of DNA which is condensed to form a body known as the nucleoid(Fig. 1) (see Topic F2).Bacterial cellwallsTo protect the cell from mechanical injury and osmotic pressure, most prokaryotes are surrounded by a rigid 3–25 nm thick cell wall (Fig. 1). The cell wall iscomposed of peptidoglycan, a complex of oligosaccharides and proteins. Theoligosaccharide component consists of linear chains of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (NAM) linked β(1–4) (see TopicJ1). Attached via an amide bond to the lactic acid group on NAM is a D-aminoacid-containing tetrapeptide. Adjacent parallel peptidoglycan chains are covalently cross-linked through the tetrapeptide side-chains by other short peptides.The extensive cross-linking in the peptidoglycan cell wall gives it its strengthand rigidity. The presence of D-amino acids in the peptidoglycan renders the cellwall resistant to the action of proteases which act on the more commonlyOutermembraneCell wallPeriplasmic dFig. 1.Prokaryote cell structure.DNA

A1 – Prokaryote cell structure3occurring L-amino acids (see Topic B1), but provides a unique target for theaction of certain antibiotics such as penicillin. Penicillin acts by inhibiting theenzyme that forms the covalent cross-links in the peptidoglycan, thereby weakening the cell wall. The β(1–4) glycosidic linkage between NAM and GlcNAc issusceptible to hydrolysis by the enzyme lysozyme which is present in tears,mucus and other body secretions.Bacteria can be classified as either Gram-positive or Gram-negativedepending on whether or not they take up the Gram stain. Gram-positivebacteria (e.g. Bacillus polymyxa) have a thick (25 nm) cell wall surrounding theirplasma membrane, whereas Gram-negative bacteria (e.g. Escherichia coli) have athinner (3 nm) cell wall and a second outer membrane (Fig. 2). In contrast withthe plasma membrane, this outer membrane is very permeable to the passage ofrelatively large molecules (molecular weight 1000 Da) due to porin proteinswhich form pores in the lipid bilayer. Between the outer membrane and the cellwall is the periplasm, a space occupied by proteins secreted from the cell.Bacterial flagellaMany bacterial cells have one or more tail-like appendages known as flagella.By rotating their flagella, bacteria can move through the extracellular mediumtowards attractants and away from repellents, so called chemotaxis. Bacterialflagella are different from eukaryotic cilia and flagella in two ways: (1) eachbacterial flagellum is made of the protein flagellin (53 kDa subunit) as opposedto tubulin (see Topic A3); and (2) it rotates rather than bends. An E. colibacterium has about six flagella that emerge from random positions on thesurface of the cell. Flagella are thin helical filaments, 15 nm in diameter and 10μm long. Electron microscopy has revealed that the flagellar filament contains11 subunits in two helical turns which, when viewed end-on, has the appearanceof an 11-bladed propeller with a hollow central core. Flagella grow by the addition of new flagellin subunits to the end away from the cell, with the newsubunits diffusing through the central core. Between the flagellar filament andthe cell membrane is the flagellar hook composed of subunits of the 42 kDahook protein that forms a short, curved structure. Situated in the plasmamembrane is the basal body or flagellar motor, an intricate assembly of proteins.The flexible hook is attached to a series of protein rings which are embedded inthe inner and outer membranes. The rotation of the flagella is driven by a flowof protons through an outer ring of proteins, called the stator. A similar protondriven motor is found in the F1F0-ATPase that synthesizes ATP (see Topic L2).(b)(a)PlasmamembraneFig. 2.Peptidoglycancell wallPeriplasmicspaceOutermembranePlasmame

BIOS INSTANT NOTES Series Editor: B.D. Hames, School of Biochemistry and Microbiology, University of Leeds, Leeds, UK Biology Animal Biology, Second Edition Biochemistry, Third Edition Bioinformatics Chemistry for Biologists, Second Edition Developmental Biology Ecology, Second Edition Genetics, Second Edition Immunology, Second Edition