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BIOCHEMISTRYl ; i) i K I ii H l) i i i o \
GEOFFREY ZUBAYColumbia University
Technische Hochschule DarmstadtFACHBEREICH 10 - BiOLOGIE
- B i b l i o t h e k -SchnittspahnstraBe 10
D-64287 Darmstadt
Mfl
Wm. C. Brown PublishersDubuque, IA Bogota Boston Buenos Aires Caracas ChicagoGuilford, CT London Madrid Mexico City Sydney Toronto
BRIEF CONTENTS
P "A R T
AN OVERVIEW OF BIOCHEMISTRY AND
BlOENERGETICS 1
Cells, Organelles, and Biomolecules 3Thermodynamics in Biocherhistry 27
CHAPTER 1
CHAPTER 2
P A R T
CHAPTER 3
CHAPTER 4
CHAPTER 5
CHAPTER 6
CHAPTER 7
P A R T
PROTEIN STRUCTURE AND FUNCTION 4 3
The Structure and Function ofWater 45The Building Blocks of Proteins:Amino Acids, Peptides, andPolypeptides 60The Three-Dimensional Structures ofProteins 79Functional Diversity of Proteins 115Methods for Characterization andPurification of Proteins 140
CATALYSIS 157
Enzyme Kinetics 159Mechanisms of Enzyme Catalysis 177Regulation of Enzyme Activities 214Vitamins and Coenzymes 237
CHAPTER 8
CHAPTER 9
CHAPTER 10
CHAPTER 11
P A R T
CHAPTER 12
CHAPTER 13
CHAPTER 14
CHAPTER 15
CHAPTER 16
METABOLISM OF CARBOHYDRATES 265
Metabolic Strategies 267Structures of Sugars and Energy-Storage Polysaccharides 282Glycolysis, Gluconeogenesis, and thePentose Phosphate Pathway 293The Tricarboxylic Acid Cycle 324Electron Transport, ProtonTranslocation, and OxidativePhosphorylation 346
CHAPTER 17
CHAPTER 18
P A R T
Photosynthesis and Other ProcessesInvolving Light 371Structures and Metabolism ofPolysaccharides andGlycoproteins 402
CHAPTER 19
CHAPTER 20
CHAPTER 21
CHAPTER 22
CHAPTER 23
P A R T
METABOLISM OF LIPIDS 4 4 1
Lipids and Membranes 443Mechanisms of MembraneTransport 462Metabolism of Fatty Acids 479Biosynthesis of Membrane LipidsMetabolism of Cholesterol 532
507
CHAPTER 24
CHAPTER 25
CHAPTER 26
CHAPTER 27
METABOLISM OF NITROGEN-CONTAINING
COMPOUNDS 561
Amino Acid Biosynthesis and Nitrogei ;Fixation in Plants andMicroorganisms 563Amino Acid Metabolism inVertebrates 597Nucleotides 629Integration of Metabolism in
.
CHAPTER
CHAPTER
P A R
2829
T
Vertebrates 666NeurotransmissionVision 717
8
698
CHAPTER 30
CHAPTER 31
CHAPTER 32
CHAPTER 3"3
STORAGE AND UTILIZATION OF GENETIC
INFORMATION 731
Structures of Nucleic Acids andNucleoproteins 733DNA Replication, Repair, andRecombination 760DNA Manipulation and ItsApplications 790RNA Synthesis and Processing 818
VI
CHAPTER 34 Protein Synthesis, Targeting, andTurnover 849
CHAPTER 35 Regulation of Gene Expression inProkaryotes 883
CHAPTER 36 Regulation of Gene Expression inEukaryotes 913
CHAPTER 37 Immunobiology 944CHAPTER 38 Cancer and Carcinogenesis 963CHAPTER 39 The Human Immunodeficiency Virus
(HIV) and Acquired ImmunodeficiencySyndrome (AIDS) 979
Brief Contents VH
CONTENTS
P -A R T
F- -T-Vift •%' ! AN OVERVIEW OF BIOCHEMISTRY
\"'jp
AND BlOENERGETICS 1
CHAPTER
Cells, Organelles, and Biomolecules 3Geoffrey Zubay
All Organisms Are Composed of Cells 3Cells Are Composed of Small Molecules, Macromolecules,
and Organelles _-5Macromolecules Conceal Their Hydrophobic Parts 8Biochemical Reactions Form a Small Subset of Ordinary
Chemical Reactions 12Biochemical Reactions Occur under Mild ConditionsMany Biochemical Reactions Require Energy 16Biochemical Reactions Are Localized in the Cell 18Biochemical Reactions Are Organized into PathwaysBiochemical Reactions Are Regulated 19Organisms Are Biochemically Dependent on One
Another 20Information for the Synthesis of Proteins Is Carried by the
DNA 20The First Living Systems Were Acellular 21
- All Living Systems Are Related through a CommonEvolution 23
16
19
CHAPTER
Thermodynamics in Biochemistry 27Geoffrey Zubay
Thermodynamic Quantities 28The First Law of Thermodynamics: Any Change in the
Energy of a System Requires an Equal and Opposite <.Change in the Surroundings 28
The Second Law of Thermodynamics: In AnySpontaneous Process the Total EntropyIncreases 29
Free Energy Provides the Most Useful Criterion forSpontaneity 32
Applications of the Free Energy Function 33Values of Free Energy Are Known for Many
Compounds 33
The Standard Free Energy Change in a Reaction IsRelated Logarithmically to the EquilibriumConstant 33
Free Energy Is the Maximum Energy Available forUseful Work 35
Biological Systems Perform Various Kinds of Work 351,Favorable Reactions Can Drive Unfavorable ;
Reactions 35ATP as the Main Carrier of Free Energy in Biochemical '
Systems 36 ;The Hydrolysis of ATP Yields a Large Amount of Free '
Energy 36 ':
P A R T
PROTEIN STRUCTURE AND
FUNCTION 4 3
CHAPTER
The Structure and Function of Water 45 !Geoffrey Zubay
Liquid Water and Ice Have Very Similar Structures 45A Variety of Forces Affect the Interactions between
Biomolecules and Water 48Electrostatic Forces Favor Interaction between Water,
Charged Molecules, and Polar Molecules 48Van der Waals Forces Are of Two Types 48Hydrogen Bond Forces Involve Interactions with
Unshielded Protons 49Hydrophobic Forces Are Primarily Due to Entropic
Factors 50Solubility and Related Phenomena Are Best Considered in
Thermodynamic Terms 50The Hydrogen Ion Concentration Has a Major Impact on
Biomolecular Reactions . 51The Hydrogen and Hydroxide Ion Concentrations in
Liquid Water Are Reciprocally Related 51The Extent of Ionization of a Weak Acid in Water Is a
Function of Its Acid Dissociation Constant, Ka 52i Buffered Solutions Are Resistant to Changes in pH 5-Weak Acids Buffer the pH in the Fluid Compartments of th
^ 54The^Intracellular pH Is Buffered by Mono and
Dihydrogen Phosphates 55
vu i
The pH of the Blood Plasma Is Stabilized by a Buffer, System Involving Bicarbonate, Carbonic Acid, and
Carbon Dioxide 56Water Is Directly Involved in Many Biochemical
Reactions 58
C H A P T E R
The Building Blocks of Proteins: AminoAcids, Peptides, and Polypeptides 60Geoffrey Zubay
Amino Acids 60Amino Acids Have Both Acid and Base Properties 61
•• Aromatic Amino Acids Absorb Light in the Near-Ultraviolet 64
All Amino Acids Except Glycine Show Asymmetry 64Peptides and Polypeptides 65Determination of Amino Acid Composition of Proteins 67
'] Determination of Amino Acid Sequence of Proteins 69Chemical Synthesis of Peptides and Polypeptides 75
C H A P T E R
The Three-Dimensional Structures ofProteins 79Geoffrey Zubay
The Information for Folding Is Contained in the PrimaryStructure 80
The Ramachandran Plot Predicts Sterically PermissibleStructures 81
Protein Folding Reveals aHierarchy of StructuralOrganization 83 ~~ J
Two Secondary Structures Are Found in Most Proteins 85The a Helix 85The (3 Sheet 88
Pauling and Corey Provided the Foundation for OurUnderstanding of Fibrous Protein Structures 88
Collagen Forms a Unique Triple-Stranded Structure 90In Globular Proteins, Secondary Structure Elements Are
Connected in Simple Motifs 92The Domain Is the Basic Unit of Tertiary Structure 95
The Helix-Loop-Helix Motif Is the Basic ComponentFound in a-Domain Structures 96
a/p Domains Exploit the /3-a-/8 Motif 97Antiparallel ji Domains Show a Great Variety of
Topologies 98Some Proteins or Domains Require Additional Featurest. to Account for Their Stability 99Many Proteins Contain More Than One Domain 100
Quaternary Structure Depends on the Interaction of Two orMore Proteins or Protein Subunits 101
Predicting Protein Tertiary Structure from Protein PrimaryStructure 106
Methods for Determining Protein Conformation 107X-Ray Diffraction Analysis of Fibrous Proteins 107X-Ray Diffraction Analysis of Protein Crystals 107Nuclear Magnetic Resonance (NMR) Complements
X-Ray Crystallography 109Optical Rotatory Dispersion (ORD) and Circular
Dichroism (CD) 110
CHAPTER
Functional Diversity of Proteins 115Geoffrey Zubay
Targeting and Functional Diversity 115Proteins Are Directed to the Regions Where They Are
Utilized 115Classification of Proteins According to Location
, Emphasizes Functionality 116Protein Structure Is Suited to Protein Function 116
Hemoglobin — An Allosteric Oxygen-Binding Protein 118The Binding of Certain Factors to Hemoglobin Has a
Negative Effect on Oxygen Binding 119X-Ray Diffraction Studies Reveal Two Conformations
for Hemoglobin 122Changes in Conformation Are Initiated by Oxygen
Binding 122Two Models Have Been Proposed for the Way •
Hemoglobins and Other Allosteric ProteinsWork 128
Muscle — An Aggregate of Proteins Involved inContraction 129
Protein Diversification as a Result of EvolutionaryPressures 134
Gene Splicing Results in a Reshuffling of Domains inProteins 135
Evolutionary Diversification Is,Directly Involved inAntibody Formation 136
CHAPTER
Methods for Characterization andPurification of Proteins 140Geoffrey Zubay
Methods of Protein Characterization 140Solubility Reflects a Balance of Protein-Solvent
Interactions 140Several Methods Are Available for Determination of
' v Gross Size and Shape 141X-Electrophoretic Methods Are the Best Way to Analyze
•', Mixtures 145Methods of Protein Purification 147
Differential Centrifugation^Subdivides Crude Extractsintd'Iwo or More Fractions 148
Differentiah-Precipitation Is Based on SolubilityDifferences ^148
Contents IX
Column Procedures Are the Most Versatile PurificationMethods 148
Electrophoretic Methods Are Used for Preparation andAnalysis 150
Purification of Specific Proteins Involves Combinationsof Different Procedures 150
P A R T
CATALYSIS 1 5 7
CHAPTER
Enzyme Kinetics 159Geoffrey Zubay
The Discovery of Enzymes 159Enzyme Terminology 160Basic Aspects of Chemical Kinetics 160
A Critical Amount of Energy Is Needed for theReactants to Reach the Transition State 161
Catalysts Speed up Reactions by Lowering the FreeEnergy of Activation 162
Kinetics of Enzyme-Catalyzed Reactions 163Kinetic Parameters Are Determined by Measuring the
Initial Reaction Velocity as a Function of theSubstrate Concentration 163
The Henri-Michaelis-Menten Treatment Assumes Thatthe Enzyme-Substrate Complex Is in Equilibriumwith Free Enzyme and Substrate 164
Steady-State Kinetic Analysis Assumes That theConcentration of the Enzyme-Substrate ComplexRemains Nearly Constant 165
Kinetics of Enzymatic Reactions Involving TwoSubstrates 168 )
Effects of Temperature and pH on EnzymaticActivity 169
Enzyme Inhibition 169Competitive Inhibitors Bind at the Active Site 169Noncompetitive and Uncompetitive Inhibitors Do Not
Compete Directly with Substrate Binding 171Irreversible Inhibitors Permanently Alter the Enzyme
Structure 171
CHAPTER
Mechanisms of Enzyme Catalysis 177Geoffrey Zubay
Five Themes That Recur in Discussing EnzymaticReactions 177
The Proximity Effect: Enzymes Bring Reacting SpeciesClose Together 178
General-Base and General-Acid Catalysis Provide \-Ways of Avoiding the Need for Extremely High or FLowpH 178 \
Electrostatic Interactions Can Promote the Formation)of the Transition State 180 ;|
Enzymatic Functional Groups Provide Nucleophilic at,Electrophilic Catalysts 181 '
Structural Flexibility Can Increase the Specificity of ••Enzymes 182
Detailed Mechanisms of Enzyme Catalysis 183 ;.Serine Proteases Are a Diverse Group of Enzymes Thf
Use a Serine Residue for Nucleophilic ,Catalysis 184
Zinc Provides an Electrophilic Center in SomeProteases 190
Ribonuclease A: An Example of Concerted Acid-BaseiCatalysis 194 :'
Triosephosphate Isomerase Has Approached CatalytiliPerfection 199
Lysozyme Hydrolyzes Complex PolysaccharidesContaining Five or More Residues 202
Lactate Dehydrogenase: A Bisubstrate Enzyme 206Binding of Coenzyme Occurs before Binding of
Sugar 207 jKinetic Studies Reveal Intermediates and Slow Step it
the Reaction 207 >Reaction Results from Concerted Catalysis 209 \Isoenzymes of Lactate Dehydrogenase Serve Different
Functions 209Alcohol Dehydrogenase Uses Zinc as an Electrophili
Catalyst 210 \
CHAPTER
Regulation of Enzyme Activities 214Geoffrey Zubay
Partial Proteolysis Results in Irreversible CovalentModifications 215
Phosphorylation, Adenylylation, and Disulfide ReductionLead to Reversible Covalent Modifications 216
Allosteric Regulation Allows an Enzyme to Be ControlledRapidly by Materials That Are Structurally Unrelated tothe Substrate 218
. Allosteric Enzymes Typically Exhibit a SigmoidalDependence on Substrate Concentration 218 |
The Symmetry Model Provides a Useful Framework /ifRelating Conformational Transitions to Allosteric IActivation or Inhibition 220 j;
Allosteric Control of Phosphofructokinase Is Consistent wi!. the Symmetry Model 221 |<In Aspartate Carbamoyl Transferase the Catalytic and
Regulatory Sites Are Located on Different Subunits 2The Advantages of Positive Cooperativity 229Negative Cooperativity 229
Contej
Glycogen Phosphorylase Activity Is Regulated by AllostericEffectors and by Phosphorylation 229
Calmodulin Regulates Other Regulatory Proteins by Protein-Protein Interaction 233
C H A P T E R
Vitamins and Coenzymes 237Perry A. Frey
Water-Soluble Vitamins and Their Coenzymes 238Thiamine Pyrophosphate Is Involved in C—C and
C—X Bond Cleavage 238Pyridoxal-5' -Phosphate Is Required for a Variety of
Reactions with a-Amino Acids 240Nicotinamide Coenzymes Are Used in Reactions
Involving Hydride Transfers 242"Flavins Are Used in Reactions Involving One or Two
Electron Transfers 244Reactions Requiring Acyl Activation Frequently Use
Phosphopantetheine Coenzymes 247a-Lipoic Acid Is the Coenzyme of Choice for Reactions
Requiring Acyl-Group Transfers Linked toOxidation-Reduction 249
Biotin Mediates Carboxylations 250Folate Coenzymes Are Used in Reactions for One-
Carbon Transfers 251 ^Ascorbic Acid Is Required to Maintain the Enzyme
That Forms Hydroxyproline Residues inCollagen 251
Vitamin B]2 Coenzymes Are Associated withRearrangements on Adjacent Carbon Atoms 253
Iron-Containing Coenzymes Are' Frequently Involved inRedox Reactions 255
Metal Cofactors 258Lipid-Soluble Vitamins 259
P A R T
METABOLISM OF
CARBOHYDRATES 265
CHAPTER
Metabolic Strategies 267Geoffrey Zubay
Living Cells Require a Steady Supply of Starting Materialsand Energy 267
Organisms Differ in Sources of Energy, Reducing Power, andStarting Materials for Biosynthesis 267
Reactions Are Organized into Sequences "or Pathways 268Sequentially Related Enzymes Are Frequently
Clustered 269
Pathways Show Functional Coupling 270The ATP-ADP System Mediates Conversions in Both
Directions 271Conversions Are Kinetically Regulated 271Pathways Are Regulated by Controlling Amounts and
Activities of Enzymes 273Enzyme Activity Is Regulated by Interaction with
Regulatory Factors 273Regulatory Enzymes Occupy Key Positions in
Pathways 273A Regulated Reaction Is Effective Only If It Is
Exergonic 274Regulatory Enzymes Often Show Cooperative
Behavior 274Both Anabolic and Catabolic Pathways Are Regulated
by the Energy Status of the Cell 275Regulation of Pathways Involves the Interplay of
Kinetic and Thermodynamic Factors 276Strategies for Pathway Analysis 276,
Analysis of Single-Step Pathways 277Analysis of Multistep Pathways 277Radiolabeled Compounds Facilitate Pathway
Analysis 279Pathways Are Usually Studied Both in Vitro and in
Vivo 279
CHAPTER
Structures of Sugars and Energy-StoragePolysaccharides 282Pamela Stanley and Geoffrey Zubay
Monosaccharides and Related Compounds 282Families of Monosaccharides Are Structurally
Related 283Monosaccharides Cyclize to Form Hemiacetals 283Monosaccharides Are Linked by Glycosidic Bonds 286
Disaccharides and Polysaccharides 286Cellulose Is a Major Homopolymer Found in Cell'
Walls 287Starch and Glycogen Are Major Energy-Storage
Polysaccharides 289The Configurations of Glycogen and Cellulose Dictate
Their Roles 290
CHAPTER
Glycolysis, Gluconeogenesis, and thePentose Phosphate Pathway 293Geoffrey Zubay
Overview of Glycolysis 294TRree Hexose Phosphates Constitute the First
Metabolic Pool 294Phosphorylase Converts Storage Carbohydrates to
Glucose Phosphate 294
Contents xi
Hexokinase Converts Free Sugars to HexosePhosphates 297 '
Phosphoglucomutase Interconverts Glucose-1-phosphate and Glucose-6-phosphate 298
Phosphohexoisomerase Interconverts Glucoses-phosphate and Fructose-6-phosphate 298
— Formation of Fructose-1,6-bisphosphate Signals aCommitment to Glycolysis 298
Fructose-1,6-bisphosphate and the Two TriosePhosphates Constitute the Second Metabolic Pool inGlycolysis 300
Aldolase Cleaves Fructose-1,6-bisphosphate 300Triose Phosphate Isomerase Interconverts the Two
Trioses 300The Conversion of Triose Phosphates to A
Phosphoglycerates Occurs in Two Steps 300The Three-Carbon Phosphorylated Acids Constitute a
Third Metabolic Pool 302Conversion of Phosphoenolpyruvate to Pyruvate
Generates ATP 302The NAD+ Reduced in Glycolysis Must Be
Regenerated 304 sSummary of Glycolysis 304Catabolism of Other Sugars 304Fructose 305Galactose 305
Gluconeogenesis 306 r)
Gluconeogenesis Consumes ATP 306Conversion of Pyruvate to Phosphoenolpyruvate
Requires Two High-Energy Phosphates 306Conversion of Phosphoenolpyruvate to Fructose-1,6-
bisphosphate Uses the Same Enzymes asGlycolysis 309
Fructose-bisphosphate Phosphatase Converts Fructose-1,6-bisphosphate to Fructose-6-phosphate 309
Hexose Phosphates Can Be Converted to StoragePolysaccharides 309
Summary of Gluconeogenesis 311Regulation of Glycolysis and Gluconeogenesis 311
How Do Intracellular Signals Regulate EnergyMetabolism? 313
Hormonal Controls Can Override IntracellularControls 313
Hormonal Effects of Glue agon Are Mediated by CyclicAMP 313
The Hormone Epinephrine Stimulates Glycogenolysisin Both Liver Cells and Muscle Cells 314 <*
Hormonal'Regulation of the Flux between Fructose-6- \phosphate and Fructose-1,6-bisphosphate in the >.Liver Is Mediated by Fructose-2,6-bisphosphate 315
Summary of the Regulation of Glycolysis andGluconeogenesis 316 J
The Pentose Phosphate Pathway,, 316Two NADPH Molecules Are Generated by the Pentose
Phosphate Pathway 317
Transaldolase and Transketolase Catalyze theInterconversion of Many PhosphorylatedSugars 317
Production of Ribose-5-phosphate and Xylulose-5-phosphate 318
CHAPTER
The Tricarboxylic Acid Cycle 324 ,Geoffrey Zubay
iDiscovery of the TCA Cycle 325 .j
Steps in the TCA Cycle 327 ::The Oxidative Decarboxylation of Pyruvate Leads to
Acetyl-CoA 327 >•Citrate Synthase Is the Gateway to the TCA Cycle 3'iAconitase Catalyzes the Isomerization of Citrate to i
Isocitrate ~330 IIsocitrate Dehydrogenase Catalyzes the First Oxidatiol' in the TCA Cycle 331 j;a-Ketoglutarate Dehydrogenase Catalyzes the ;!
Decarboxylation of a-Ketoglutarate to Succinyl- Ii CoA 332 f
Succinate Thiokinase Couples the Conversion ofSuccinyl-CoA to Succinate with the Synthesis ofGTP 332
Succinate Dehydrogenase Catalyzes the Oxidation ofSuccinate to Fumarate 332 j
Fumarase Catalyzes the Addition of Water to Fumarmto Form Malate 333 I
Malate Dehydrogenase Catalyzes the Oxidation of |Malate to Oxaloacetate 333 j
Stereochemical Aspects of TCA Cycle Reactions 333 |ATP Stoichiometry of the TCA Cycle 333 jThermodynamics of the TCA Cycle 335The Amphibolic Nature of the TCA Cycle 335The Glyoxylate Cycle Permits Growth of a Two-Carbon
Source 336Utilization ofjhe Succinate Requires Passage from th\
Glyoxysome to the Mitochondria 339 |jOxidation of Other Substrates by the TCA Cycle 340 |The TCA Cycle Activity Is Regulated at Metabolic S
Branchpoints 340 EThe Pyruvate Branchpoint Partitions Pyruvate betwee\
Acetyl-CoA and Oxaloacetate 340 J«Citrate Synthase Is Negatively Regulated by NADH an
the Energy Charge 342 |Isocitrate Dehydrogenase Is Regulated by the NADH-i^ tp-NAD+ Ratio and the Energy Charge 343 f
a-Ketoglutarate Dehydrogenase Is Negatively I?Regulated by NADH 343 i|
xii Conte'i
CHAPTER CHAPTER
Electron Transport, Proton Translocation,and Oxidative Phosphorylation 346Geoffrey Zubay
Electron Transport Is a Membrane-Localized Process 347A Bucket Brigade of Molecules Carries Electrons from
the TCA Cycle to O2 347The Sequence of Electron Carriers Was Deduced from
Kinetic Measurements 350Redox Potentials Give a Measure of Oxidizing and
Reducing Strengths of the Different ElectronCarriers 350
Most of the Electron Carriers Exist in LargeComplexes 351
The Main Function of the Mitochondrial Electron >Transport Complexes Is to Translocate Protons -353
Complexes I and II Mediate the Transfer of Electronsfrom NADH and FADH2 to Ubiquinone 354
Complex III, the Cytochrome bcj Complex, TransfersElectrons from QH2 to Cytochrome WhileTranslocating Protons by a Redox Loop 355
Complex IV, the Cytochrome Oxidase Complex,Transfers Electrons from jCytochrome c to O2 WhilePumping Protons across the Membrane 355
Reconstitution Experiments Demonstrate the Key Rolesof Ubiquinone and Cytochromer c as Mobile ElectronCarriers between the Giant Complexes 356
Experiments on Mitochondrial SuspensionsDemonstrate That Electron Transport Creates anElectrochemical Potential Gradient for Protonsacross the Inner Membrane 357
Oxidative Phosphorylation 359^The Respiratory Chain Contains Three Coupling Sites
for ATP Formation 359Electron Transfer Is Tightly Coupled to ATP
Formation 359The Chemiosmotic Theory Proposes That
Phosphorylation Is Driven by ProtonMovements 360
Flow of Protons Back into the Matrix Drives theFormation of ATP 361
The Proton-Conducting ATP-Synthase or ATPase: Fjand Fo 362
The Mechanism of Action of the ATP-Synthase 362Transport of Substrates, Pj, ADP, and ATP into and out of
Mitochondria 365Uptake of Pt and Oxidizable Substrates Is Coupled to
the Release of OH~ Ions 365^Export of ATP Is Coupled to ADP Uptake 366
Electrons from Cytosolic NADH Are Imported byShuttle Systems 366
Complete Oxidation of Glucose Yields about 30Molecules of ATP 367
Photosynthesis and Other ProcessesInvolving Light 371Geoffrey Zubay-
Photosynthesis. ,371The Photochemical Reactions of Photosynthesis Take
Place in Membranes 373Photosynthesis Depends on the Photochemical
Reactivity of Chlorophyll 374Photooxidation of Chlorophyll Generates a Cationic
Free Radical 378The Reactive Chlorophyll Is Bound to Proteins in
Complexes Called Reaction Centers 379In Purple Bacterial Reaction Centers, Electrons Move
from P870 to Bacteriopheophytin and Then toQuinones 380
A Cyclic Electron-Transport Chain Returns'Electronsto P870 and Moves Protons Outward across theMembrane; Flow of Protons Back into the CellDrives the Formation of ATP 381
An Antenna System Transfers Energy to the ReactionCenters 382
Chloroplasts Have Two Photosystems Linked inSeries 385 -
Photosystem I Reduces NADP+ by Way of Iron-SulfurProteins 389
02 Evolution Requires the Accumulation of FourOxidizing Equivalents in the Reaction Center ofPhotosystem II 390
Flow of Electrons from H2O to NADP+ Drives ProtonTransport into the Thylakoid Lumen; Protons Returnto the Stroma through an ATP-Synthase 391
Carbon Fixation: The Reductive Pentose Cycle 393Ribulose Bisphosphate Carboxylase/Oxygenase,
Photorespiration, and the C4 Cycle 393Other Biochemical Processes Involving Light 397
Phytochrome Synchronizes Circadian and SeasonalRhythms in Plants 397
Bioluminescence 397
CHAPTER
Structures and Metabolism ofPolysaccharides and GlycoproteinsPamela Stanley
402
Monosaccharides Are Often Formed by Interconversionsbetween Hexoses 403
the Hexose Monophosphate Pool Includes Mannose asWell as Glucose and Fructose 403
GalactdseAs Not a Member of the HexoseMonophosphate Pool 403
Contents xiii
Hexose Modifications Involve Alterations or Additionsof Small Substituents 404
Disaccharide Biosynthesis 407Energy-Storage Polysaccharides Are Simple
Homopolymers 407Structural Polysaccharides Include Homopolymers and
_Heteropolymers 408Chitin Contains a Different Building Block 408 ;vHeteropolysaccharides Contain More Than One
Building Block 409Proteoglycans Are Complexes of Proteins with
Glycans 411In Glycoproteins, Oligosaccharides Are Covalently Linked to
N or O Atoms in Protein Amino Acid Side Chains 412Carbohydrate Modification Is Important in Targeting
Certain Enzymes to the Lysosomes 413A Carbohydrate-Lipid Serves to Ancho/Some
Glycoproteins to the Cell Surface 413Carbohydrates of the Plasma Membrane Are Important in
Cell Recognition 414 '"Determination of Carbohydrate Primary Structure Requires
Purification before Structural Analysis 416Oligosaccharides Are Synthesized in a Concerted Fashion by
Specific Glycosyltransferases 418Biosynthesis of N-Linked Oligosaccharides 421Biosynthesis of O-Linked Oligosaccharides 426Specific Inhibitors and Mutants Are Used to Explore
the Roles of Glycoprotein Carbohydrates 430Bacterial Cell Walls Are Composed of Polysaccharides
Cross-Linked by Peptides 430Bacterial Cell Wall Biosynthesis 431 .
Synthesis of the UDP-N-Acetylmuramyl-PentapeptideMonomer Occurs in the Cytoplasm 432
Formation of Linear Polymers of the Peptidoglycan IsMembrane-Associated 432
Cross-Linking of the Peptidoglycan Strands Occurs onthe Noncytoplasmic Side of the PlasmaMembrane 433
Penicillin Inhibits the Transpeptidation Reaction 434
P A R T
METABOLISM OF LIPIDS 441
CHAPTER
Lipids and Membranes 443Dennis E. Vance
The Structure of Biological Membranes 444Different Membrane Structures Can Be Separated According
to Their Density 444Membranes Contain Complex Mixtures of Lipids 445
Phospholipids Spontaneously Form Ordered Structures in ;!Water 448 ' \
Membranes Have Both Integral and Peripheral Proteins 45(1Integral Membrane Proteins Contain Transmembrane a
Helices 451Proteins and Lipids Can Move around within i
Membranes 453 •Biological Membranes Are Asymmetrical 455Membrane Fluidity Is Sensitive to Temperature and Lipid j
Composition 456 iSome Proteins of Eukaryotic Plasma Membranes Are
Connected to the Cytoskeleton 459
CHAPTER
Mechanisms of Membrane Transport 462Gary R. Jacobson and Geoffrey Zubay
Transport of Materials across Membranes 462Most Solutes Are Transported by Specific
Carriers 462Some Transporters Facilitate Diffusion of a Solute
down an Electrochemical Potential Gradient 464Active Transport against-an Electrochemical Potential
Gradient Requires Energy 464Isotopes, Substrate Analogs, Membrane Vesicles, and ;j
Bacterial Mutants Are Used to Study Transport 46.Molecular Models of Transport Mechanisms 467 fThe Catalytic Cycle of the Na+-K+ Pump Includes Tw{
Phosphorylated Forms of the Enzyme 468Some Membranes Have Relatively Large Pores 469Vesicular Transport 470
Hormone Receptors and Enzymes in Membranes TransportSignals 471
Many Hormone Receptors Trigger G Proteins toActivate'or Inhibit Adehylate Cyclase 471
The Receptors for Insulin and Some Growth FactorsAre Tyrosine Kinases 474
Other Receptors Trigger Breakdown ofPhosphatidylinositol to Inositol Trisphosphate andDiacylglycerol 475
|!CHAPTER
Metabolism of Fatty Acids 479 I'Dennis E. Vance „ s'
Fatty Acid Degradation 479 •'Fatty Acids Originate from Three Sources: Diet, 'i
Adipocytes, and de novo Synthesis 479 s
Fatty Acid Breakdown Occurs in Blocks of Two Carbd.Atoms 480 "
:%. The Oxidation of Saturated Fatty Acids Occurs in . !:*X^ Mitochondria 482 f
Fatty. Acid Oxidation Yields Large Amounts of \,A7K 482 '• '•'•'
xiv Conteii
Additional Enzymes Are Required for Oxidation ofUnsaturated Fatty Acids in Mitochondria 484
Fatty Acids with an Odd Number of Carbons AreOxidized to Propionyl-CoA 484
Fatty Acids Can Also Be Oxidized by a or wOxidation 486
Ketone Bodies Formed in the Liver Are Used forEnergy in Other Tissues 487
[3 Oxidation Also Occurs in Peroxisomes 488Summary of Fatty Acid Degradation 488
Biosynthesis of Saturated Fatty Acids 489The,First Step in Fatty Acid Synthesis Is Catalyzed by
Acetyl-CoA Carboxylase 489Seven Reactions Are Catalyzed by the Fatty Acid
Synthase 490The Organization of the Fatty Acid Synthase Is
Different in E. coli and Animals 491Biosynthesis of Monounsaturated Fatty Acids Follows
Distinct Routes in E. coli and Animal Cells 491Biosynthesis of Polyunsaturated Fatty Acids Occurs
Mainly in Eukaryotes 496Summary of the Pathways for Synthesis and
Degradation 497Regulation of Fatty Acid Metabolism 497
The Release of Fatty Acids from Adipose Tissue IsRegulated 497
Fatty Acid-Binding Proteins and Acyl-CoA-BindingProtein May Be Important in the IntracellularTrafficking of Fatty Acids 498
Transport of Fatty Acids into Mitochondria IsRegulated 500 %
Fatty Acid Biosynthesis Is Limited by SubstrateSupply 501 •'• r'r.
Fatty Acid Synthesis Is Regulated by the First Step inthe Pathway 501 !
The Controls for Fatty Acid Metabolism DiscourageSimultaneous Synthesis and Breakdown 503
Long-Term Dietary Changes Lead to Adjustments in theLevel of Enzymes 503
CHAPTER
Biosynthesis of Membrane Lipids 507Dennis E. Vance
Phospholipids 507In E. coli, Phospholipid Synthesis Generates
Phosphatidylethanolamine, Phosphatidylglycerol,and Diphosphatidylglycerol 508
Phospholipid Synthesis in Eukaryotes Is More\ Complex 511
x Diacyglycerol Is the Key Intermediate in theBiosynthesis of Phosphatidylcholine andPhosphatidylethanolamine 511
. Fatty Acid Substituents at SN-1 and SN-2 Positions AreReplaceable 513
Phosphatidylinositol- 4,5-Bisphosphate, a Precursor ofSecond Messengers, Is Synthesized via CDP-Diacylglycerol 515
The Metabolism of Phosphatidylserine andPhosphatidylethanolamine Is Closely Linked 515
Biosynthesis of Alkyl and Alkenyl Ethers 515The Final Reactions for Phospholipid Biosynthesis
Occur on the Cytosolic Surface of the EndoplasmicReticulum 516
In the Liver, Regulation Gives Priority to Formation ofStructural Lipids over Energy-Storage Lipids 517
Phospholipases Degrade Phospholipids 520Sphingolipids 521
Sphingomyelin Is Formed Directly from Ceramide 521Glycosphingolipid Synthesis Also Starts from
Ceramide 521Sphingolipids Function as Structural Components, as
Specific Cell Receptors, and as Second-MessengerPrecursors 523
Defects in Sphingolipid Catabolism Are Associated withMetabolic Diseases 524 ' -
Eicosanoids Are Hormones Derived from ArachidonicAcid 525
Eicosanoid Biosynthesis 526Eicosanoids Exert Their Action Locally 527
CHAPTER
Metabolism of Cholesterol 532 'Dennis E. Vance
Biosynthesis of Cholesterol 532Mevalonate Is a Key Intermediate in Cholesterol
Biosynthesis 534The Rate of Mevalonate Synthesis Determines the Rate
of Cholesterol Biosynthesis 534It Takes Six Mevalonates and Ten Steps to Make
Lanostewl, The First Tetracyclic Intermediate 537From Lanostewl to Cholesterol Takes Another 20
Steps 538Summary of Cholesterol Biosynthesis 540
Lipoprotein Metabolism 542There Are Five Classes of Lipoproteins in Human
Plasma 542Lipoproteins Are Made in the Endoplasmic Reticulum
of the Liver and Intestine 543Chylomicrons and Very-Low-Density Lipoproteins
(VLDL) Transport Cholesterol and Triacylglycerol toOther Tissues 547
\ Low-Density Lipoproteins (LDL) Are Removed from the\, Plasma by the Liver, Adrenals, and Adipose\ Tissue 548Serious Diseases Result from Cholesterol
Deposits 549High-ueHsity Lipoproteins (HDL) May Reduce
CholestewliDeposits 550
Contents XV
Bile Acid Metabolism - 550Metabolism of Steroid Hormones 551Overview of Mammalian Cholesterol Metabolism 555
P A R T
METABOLISM OF NITROGEN-
CONTAINING COMPOUNDS 561
CHAPTER
Amino Acid Biosynthesis and NitrogenFixation in Plants and Microorganisms 563Ronald Somerville, H. Edwin Umbarger, and Geoffrey Zubay
The Pathways to Amino Acids Are Branchpoints from theCentral Metabolic Pathways 564
Our Understanding of Amino Acid Biosynthesis HasResulted from Genetic and BiochemicalInvestigations 564
The Number of Proteins Participating in a Pathway Is^Known through Genetic ComplementationAnalysis 564
Biochemists Use the Auxotrophs Isolated byy., . .'-'Geneticists '564
The Glutamate Family of Amino Acids and NitrogenFixation 564
The Direct Amination of a-Ketoglutarate Leads toGlutamate 565
Amidation of Glutamate to Glutamine Is an ElaboratelyRegulated Process 567
The Nitrogen Cycle Encompasses a Series of Reactionsin. Which Nitrogen Passes through Many Forms. 569
Three Enzymes Convert Glutamate to Proline 570Arginine Biosynthesis Uses Some Reactions Seen in the
Urea Cycle 570The Biosynthesis of Amino Acids of the Serine Family
(L-Serine, Glycine, and L-Cysteine) and the Fixation of% Sulfur 570, Three Enzymes Convert 3-Phospho-D-Glycerate to
Serine 572Two More Enzymes Convert L-Serine to Glycine 572Cysteine Biosynthesis Involves Sulfhydryl Transfer to
• Activated Serine 572Sulfate Must Be Reduced to Sulfide before \
Incorporation into Amino Acids 574 vThe Biosynthesis of Some Amino Acids of the Aspartate \.\
Family: L-Aspartate, L-Asparagine, L-Methionine, and ';1 L-Threonine 575
Aspartate Is Formed from Oxaloacetate in aTransamination Reaction 576
Asparagine Biosynthesis Requires ATP andGlutamine 576
L-Aspartic-fl-Semialdehyde Is a Common ^in L-Lysine, L-Methionine, and L-Threonine ':Synthesis 576 ,',
Methionine Is Important as a Protein Constituteas a Precursor of Other Cell Components viS-Adenosylmethionine 577 •
The Carbon Flow in the Aspartate Family Is Rtat the Aspartokinase Step 577
The Biosynthesis of Amino Acids of the Pyruvate F;L-Alanine, L-Valine, and L-Leucine 581
L-Alanine Is Formed from Pyruvate in aTransamination Reaction 581
Isoleucine and Valine Biosynthesis Share FourEnzymes 581
L-Leucine Is Formed from a-Ketoisovalerate in)Steps 581 I
Amino Acid Pathways Absent in Mammals Off\Targets for Safe Herbicides 581 |
In the Biosynthesis of the Aromatic Family of Aminji(L-Tryptophan, L-Phenylalanine, and L-Tyrosine), jChorismate Is a Key Intermediate 584 |; Prephenate Is a Common Intermediate in L- j:
Phenylalanine and L-Tyrosine Synthesis 5$Tryptophan Is Synthesized in Five Steps from |
Chorismate 586 jjCarbon Flow in the Biosynthesis of Aromatic /
Acids Is Regulated at Branchpoints 589Histidine Constitutes a Family of One 589
- Nonprotein Amino Acids Are Derived from Protein !Acids 592 j
A Wide Variety of D-Amino Acids Are Found inMicrobes 592 J-
There Are Hundreds of Naturally Occurring A,Acid Analogs 592
CHAPTER
Amino Acid Metabolism inVertebrates 597 jRonald Somerville, H. Edwin Umbarger, and Geoffn
Humans and Rodents Synthesize Less Than Half ofAmino Acids They Need for Protein Synthesis !
Many Amino Acids Are Required in the Diet for GcNutrition 598 \
Essential Amino Acids Must Be Obtained by DegraljIngested Proteins 599 f
Amino Acids May Be Reutilized or They May Be DjWhen Present in Excess 600
Transamination Is the Most Widespread FormNitrogen Transfer 600
> Net Deamination via Transamination Requires;"'* v Oxidative Deamination 600 i
i 9
XVI
In Many Vertebrates, Ammonia Resulting from DeaminationMust Be Detoxified Prior to Elimination 602
Urea Formation Is a Complex and Costly Mode ofAmmonia Detoxification 603
The Urea Cycle and the TCA Cycle Are Linked by theKrebs Bicycle 603
More Than One Carrier Exists for TransportingAmmonia from the Muscle to the Liver 603
Amino Acid Catabolism Can Serve as a Major Source ofCarbon Skeletons and Energy 606
For Many Genetic Diseases the Defect Is in Amino AcidCatabolism 606
Most Human Genetic Diseases Associated with AminoAcid Metabolism Are Due to* Defects in TheirCatabolism 609
Amino Acids Serve as the Precursors for Compounds OtherThan Proteins 609
Porphyrin Biosynthesis Starts with the Condensation ofGlycine and Succinyl-CoA 609
Glutathione Is a Multipurpose Reducing Agent 609
Pyrimidines Are Catabolized to /3-Alanine, NHj, andCO2 654
Regulation of Nucleotide Metabolism 655Purine Biosynthesis Is Regulated at Two Levels 655Pyrimidine Biosynthesis Is Regulated at the Level of
Formation of Carbamoyl Phosphate (Eukaryotes) orCarbamoyl Aspartate (Bacteria) 657
Deoxyribonucleotide Synthesis Is Regulated by BothActivators and Inhibitors 658
Enzyme Synthesis Also Contributes to Regulation ofDeoxyribonucleotides during the Cell Cycle 658
Metabolites Are Channeled along the NucleotideBiosynthesis Pathways 658
Intracellular Concentrations of Ribonucleotides AreMuch Higher Than Those ofDeoxyribonucleotides 659
T4 Bacteriophage Infection Stimulates NucleotideMetabolism 660 -*
Biosynthesis of Nucleotide Coenzymes 661
C H A P T E R C H A P T E R
Nucleotides 629Raymond Blakley
Nucleotide Components: A Phosphoryl Group, a Pentose,and a Base 631
Overview of Nucleotide Metabolism 634Synthesis of Purine Ribonucleotides de Novo 634
Inosine Monophosphate (IMP) Is the First PurineNucleotide Formed 637
IMP Is Converted into AMP and GMP 637Synthesis of Pyrimidine Ribonucleotides de Novo 639
UMP Is a Precursor of Other PyrimidineMononucleotides 639
CTP Is Formed from UTP 640Formation of Deoxyribonucleotides by Reduction of
Ribonucleotides 642Thymidylate Is Formed from dUMP 643
Formation of Nucleotides from Bases and Nucleosides(Salvage Pathways) 645
Purine Phosphoribosyltransferases Convert Purines toNucleotides 645
Salvage of Pyrimidines Is Less Important for Mammalsand Goes through Nucleosides 646
Conversion of Nucleoside Monophosphates toTriphosphates Goes through Diphosphates 647
Inhibitors of Nucleotide Synthesis and Their Role inChemotherapy 648
Catabojism of Nucleotides 652Intracellular Catabolism of Nucleotides Is Highly
Regulated 653. Purines Are Catabolized to Uric Acid and Then to
Other Products 654
Integration of Metabolism inVertebrates 666Geoffrey Zubay
Tissues Store Biochemical Energy in Three MajorForms 666
Each Tissue Makes Characteristic Demands andContributions to the Energy Pool 667
Brain Tissue Makes No Contributions to the FuelNeeds of the Organisms 667
Heart Muscle Utilizes Fatty Acids in Preference toGlucose to Fulfill Its Energy Needs 667
Skeletal Muscle Can Function Aerobically orAnaerobically 667
Adipose Tissue Maintains Vast Fuel Reserves in theForm of Triacylglycerols 668
The Liver Is the Central Clearing House for AllEnergy-Related Metabolism 668
Pancreatic Hormones Play a Major Role inMaintaining Blood Glucose Levels 669
General Aspects of Cell Signaling 672Hormones Are Major Vehicles for Intercellular
Communication 673Hormones Are Synthesized and Secreted by Specialized
Endocrine Glands 673Polypeptide Hormones Are Stored in Secretory\ Granules after Synthesis 673Thyroid Hormones and Epinephrine Are Amino Acid
Derivatives 676Steroid fiormones Are Derived from Cholesterol 677
The Circulating^Hormone Concentration Is Regulated 681Hormone Action Is Mediated by Receptors 681
Contents xvii
Many Plasma Membrane Receptors Generate aDiffusible Intracellular Signal 682
The Adenylate Cyclase Pathway Is Triggered by aMembrane-Bound Receptor 682
Protein Phosphorylation Is the Most Common Way inWhich Regulatory Proteins Respond to HormonalSignals 682
Variability in G Proteins Adds to the Variability of theHormone-Triggered Response 684
Multicomponent Hormonal Systems Facilitate a GreatVariety of Responses 684
The Guanylate Cyclase Pathway 685Guanylyl Cyclase Can Be Activated by a Gas 685Calcium and the Inositol Trisphosphate Pathway 685Steroid Receptors Modulate the Rate of
Transcription 686Hormones Are Organized into a Hierarchy 686 •-'Diseases Associated with the Endocrine System 689
Overproduction of Hormones Is Commonly Caused byTumor Formation 689
Underproduction of Hormones Has MultipleCauses 689
"Target-Cell Insensitivity Results from a Lack ofFunctional Receptors 690
Growth Factors Are Proteins That Behave LikeHormones 690
Plant Hormones 692 :
C H A P T E R
Neurotransmission 698Gary R. Jacob son and Geoffrey Zubay
Nerve-Impulse Propagation 698An Unequal Distribution of Ionic Species Results in a
Resting Transmembrane Potential 699An Action Potential Is the Transient Change in
Membrane Potential Occurring during NerveStimulation 700
Gated Ion Channels 701Separate Channels for Na+ and K+ Have Been Found
in Excitable Cell Membranes 702The Gating Properties of Ion Channels 703Further Evidence Concerning the Structure and
Function of Ion Channels 704Synaptic Transmission: A Chemical Mechanism for
Communication between Nerve Cell and Target Cell -*707Acetylcholine Is a Common Chemical
Neurotransmitter 707 \A Number of Other Compounds Also Serve as \.
Neurotransmitters 710The Acetylcholine Receptor Is the Best-Understood
Neurotransmitter Receptor 711Synaptic Receptors Coupled to G Proteins Produce
Slow Synaptic Responses 714
Synaptic Plasticity and Learning 714Excitability Is Found in Many Different Cell
Types 715
C H A P T E R
Vision 717Geoffrey Zubay
The Visual Pigments Found in Rod and Cone Cells 1Rhodopsin Consists of 11-cis-Retinal Bound to 4
Protein, Opsin 718Light Isomerizes the Retinal of Rhodopsin to All-
trans 719 \N Transformations of Rhodopsin Can Be Detected^
Changes in Its Absorption Spectrum 719 !'Isomerization of the Retinal Causes Other Strucs
Changes in the Protein 721 !'The Conductivity Change That Results from Absorptiii
Proton 723 ;The Effect of Light Is Mediated by Guanine \
Nucleotides 724 IRegeneration of 11-cis-Retinal by Way of a Retinyl ];
Ester 726 ijRhodopsin Movement in the Disk Membrane 726 j|Bacteriorhodopsin: A Bacterial Pigment-Protein Compt
That Resembles Rhodopsin 728 j
P A R T
STORAGE AND UTILIZATION G
GENETIC INFORMATION 73
CHAPTER
Structures of Nucleic Acids andNucleoproteins 733Geoffrey Zubay
The Genetic Significance of Nucleic Acids 733Transformation Is DNA-Mediated 734 jjStudies on Viruses Confirm the Genetic Nature dl
Nucleic Acid 734 j>Structural Properties of DNA 736 j,
The Poly nucleotide Chain Contains Mononucled%Linked by Phosphodiester Bonds 738 i;
Most DNAs Exist as Double-Helix (Duplex) fi,>. Structures 739 '[
^ - Hydrogen Bonds and Stacking Forces Stabilize tlj% ••.Double Helix 740 \:
XVUl
Conformational Variants of the Double-HelixStructure 741
Helical Structures That Use Additional Kinds ofHydrogen Bonding 746
Duplex Structures Can Form Supercoils 747DNA Denaturation Involves Separation of
Complementary Strands 750DNA Renaturation Involves Duplex Formation from
Single Strands 752Chromosome Structure 754
Physical Structure of the Bacterial Chromosome 754The Genetic Map of Escherichia coli 755Eukaryotic DNA Is Complexed with Histones 755Organization of Genes within Eukaryotic
Chromosomes 756,
CHAPTER
DNA Replication, Repair, andRecombination 760Geoffrey Zubay
760The Universality of Semiconservative Replication'Overview of DNA Replication in Bacteria 763
Growth during Replication Is Bidirectional 763• Growth at the Replication Forks Is Discontinuous 764
Proteins Involved in DNA Replication 766, Characterization of DNA Polymerase I in Vitro 767
Crystallography Combined with Genetics to Produce aDetailed Picture of DNA Poll Function 767
Establishing the Normal Roles of DNA Polymerases Iand III 768
Other Proteins Required for DNA Synthesis inEscherichia coli 769
Replication of the Escherichia coli Chromosome 771Initiation and Termination of Escherichia coli
Chromosomal Replication 771Synthesis May Take Place Concurrently on Both
Strands 772DNA Replication in Eukaryotic Cells 773
Eukaryotic Chromosomal DNA 773SV40 Is Similar to Its Host in Its Mode of
Replication 774Initiation of Chromosomal Replication in
Eukaryotes 775Mitochondrial DNA Replicates Continuously on Both
Strands 776Several Systems Exist for DNA Repair 776
The Mismatch Repair System Is Important forMaintaining Genetic Stability 778
4 Synthesis of Repair Proteins Is Regulated 779DNA«Recombination 779
Enzymes Have Been Found in Escherichia coli ThatMediate the Recombination Process 780
• Other Types of Recombination 783RNA-Directed DNA Polymerases 783
Retroviruses Are RNA Viruses That Replicate through aDNA Intermediate 783
Hepatitis B Virus Is a DNA Virus That Replicatesthrough an RNA Intermediate 783
Some Transposable Genetic Elements Encode aReverse Transcriptase That Is Crucial to theTransposition Process 783
Bacterial Reverse Transcriptase Catalyzes Synthesis ofa DNA-RNA Molecule 784
Telomerase Facilitates Replication at the Ends ofEukaryotic Chromosomes 784
Other Enyzmes That Act on DNA 785
C H A P T E R
DNA Manipulation and ItsApplications 790Geoffrey Zubay
Sequencing DNA 791Methods for Amplification of Select Segments of DNA 791Amplification by the Polymerase Chain Reaction 791DNA Cloning 791
Restriction Enzymes Are Used to Cut DNA into Weil-Defined Fragments 792
Plasmids Are Used as Vectors to Clone Small Pieces ofDNA 973
Bacteriophage Vectors Are Useful for Cloning DNASegments of up to 24 kb 794
Cosmids Are Used to Clone Segments of DNA between25 kb and 50 kb in Length 795
Shuttle Vectors Can Be Cloned into Cells of DifferentSpecies 795
Constructing a "Library" 796A Genomic DNA Library Contains Clones with
Different Genomic Fragments 797A cDNA Library Contains Clones Reflecting the mRNA
Sequences 797Numerous Approaches Can Be Used to Pick the
Correct Clone from a Library 800Cloning in Systems Other Than Escherichia coli 900
Yeast Artificial Chromosomes Are Used for CloningDNA Fragments as Large as 500 kb in Length 800
Studies on Cloned Genes in Mammals Start with TissueCulture Cells 801
Oncogenes Can Be Selected from a Genomic Libraryby Subculture Cloning 803
Cloning in Plants Has Been Accomplished with aBacterial Plasmid 803
SiteVDirected Mutagenesis Permits the Restructuring ofExisting Genes 803
^Targeted Gene Replacement in Mammalian Cells 806Recombinant DNA Techniques Were Used to Characterize
the Glob'in Gene Family 807DNA Sequence Differences Were Used to Detect
Defective Hemoglobin Genes 807
Contents XIX
The (3-Globin cDNA Probe Was Used to Characterizethe Normal (3-Globin Gene 809
Chromosome Walking Permitted Identification andIsolation of the Regions around the Adult [3-GlobinGenes 810
Walking and Jumping Were Both Used to Map theCystic Fibrosis Gene 811
Will Nucleic Acids Ever Become Useful TherapeuticAgents? 814
C H A P T E R
RNA Synthesis and Processing 818Geoffrey Zubay
The First RNA Polymerase to Be Discovered Did NotRequire a DNA Template 819
DNA-RNA Hybrid Duplexes Suggest That RNA Carries theDNA Sequences 819
There Are Three Major Classes of RNA 819Messenger RNA Carries the Information for
Polypeptide Synthesis 819Transfer RNA Carries Amino Acids to the Template for
Protein Synthesis 819Ribosomal RNA Is an Integral Part of the
Ribosome 821The Fine Structure of the Ribosome Is Beginning to
Emerge 823Overview of the Transcription Process 824
Bacterial RNA Polymerase Contains FiveSubmits 824
Binding at Promoters 825Initiation at Promoters 827Alternative Sigma Factors Trigger Initiation of -,
Transcription at Promoters with Different ConsensusSequences 828
Elongation of the Transcript 828Termination of Transcription 828Comparison of Escherichia coli RNA Polymerase with
DNA Poll and PollII 828Important Differences Exist between Eukaryotic and
Prokaryotic Transcription 829Eukaryotes Have Three Nuclear RNA
Polymerases 830Eukaryotic RNA Polymerases Are Not Fully Functional
by Themselves 830Messenger RNA Transcription by Polymerase II 830The Poll Promoter Has Two Elements 832Some PollII Promoters Have Downstream
Elements 832 \In Eukaryotes, Promoter Elements Are Located at a \
Considerable Distance from the Polymerase-Binding%Site 832
Many Viruses Encode Their Own RNA Polymerases 834RNA-Dependent RNA Polymerases of RNA Viruses 835
Other Types of RNA Synthesis 836
Posttranscriptional Alterations of Transcripts 836 !Processing and Modification of tRNA Require Sev'f
Enzymes 837 j;Processing of Ribosomal Precursor Leads to Thre) \
RNAs 837 kEukaryotic Pre-mRNA Undergoes Extensive \ °
Processing 837 J \ ;
Some RNAs Are Self-Splicing 840 'Degradation of RNA by Ribonucleases 841 ;
Some Ribonucleases Are RNAs ,841 j cCatalytic RNA May Have Evolutionary Significance iRNA Editing Involves Changing Some of the Primary I.
Sequence of a Nascent Transcript 843 fInhibitors of RNA Metabolism 845 . ;"'
Some Inhibitors Act by Binding to DNA 845 'Some Inhibitors Bind to RNA Polymerase 845 j;Some Inhibitors Are Incorporated into the Grovwf
RNA Chain 845 r \.'
C H A P T E R
Protein Synthesis, Targeting, andTurnover 849Emanuel Goldman and Geoffrey Zubay
}
The Cellular Machinery of Protein Synthesis 851 \°Messenger RNA Is the Template for Protein j 0
0
Synthesis 851 jTransfer RNAs Order Activated Amino Acids on
mRNA Template 852Ribosomes Are the Site of Protein Synthesis 851 '
The Genetic Code 853 I; ;The Code Was Deciphered with the Help ofSyntt ^
Messengers 854 1'°The Code Is Highly Degenerate 855 f-*Wobble Introduces Ambiguity into Codon-Anticol °,
Interactions 856 |The Code Is Not Quite Universal 857 jjThe Rules Regarding Codon-Anticodon Pairing i %
Species-Specific 858 K 1 1The Steps in Translation 859 \ \
Synthases Attach Amino Acids to tRNAs 859 IEach Synthase Recognizes a Specific Amino Acil *
Specific Regions on Its Cognate tRNA 860 | =<Aminoacyl-tRNA Synthases Can Correct Acylati\ „•
Errors 861. \ IA Unique tRNA Initiates Protein Synthesis 86llTranslation Begins with the Binding of mRNA fqi
Ribosome 862 \, ,Dissociable Protein Factors Play Key Roles at if ...
Different Stages in Protein Synthesis on the I •„Ribosome 863 |-
s ^ w Protein Factors Aid Initiation 863 f; ',Xv. Three Elongation Reactions Are Repeated with |
^-^Incorporation of Each Amino Acid 864 i
xx
In Addition to the P Site and the A Site for BindingtRNAs the Ribosome May Possess a Third Site, the ESite 866
Two (or Three) GTPs Are Required for Each Step inElongation 867
Termination of Translation Requires Release Factorsand Termination Codons 867
Ribosomes Can Change Reading Frame duringTranslation 870
Protein Folding Is Mediated by ProteinChaperones 871
Targeting and Posttranslational Modification of Proteins 871Proteins Are Targeted to Their Destination by Signal
Sequences 873Some Mitochondrial Proteins Are Transported after
Translation 874Eukaryotic Proteins Targeted for Secretion Are
Synthesized in the Endoplasmic Reticulum 874Proteins That-Pass through the Golgi Apparatus
Become Glycosylated 875Processing of Collagen Does Not End with
Secretion 876 „;•Bacterial Protein Transport Frequently Occurs during
Translation 876Protein Turnover 876
The Lifetimes of Proteins Differ 876Abnormal Proteins Are Selectively Degraded 878Proteolytic Hydrolysis Occurs in Mammalian
Lysosomes 878Ubiquitin Tags Proteins for Proteolysis 879ATP Plays Multiple Roles in Protein Degradation 879
CHAPTER
Regulation of Gene Expression inProkaryotes 883Geoffrey Zubay
Control of Transcription Is the Dominant Mode ofRegulation in Escherichia coli 884
The Initiation Point for Transcription Is a Major Site forRegulating Gene Expression 884
Regulation of the Three-Gene Cluster Known as the LacOperon Occurs at the Transcription Level 885
fi-Galactosidase Synthesis Is Augmented by a Small-Molecule Inducer 885
A Gene Was Discovered That Leads to Repression ofSynthesis in the Absence of Inducer 887
A Locus Adjacent to the Operon Is Found to BeRequired for Repressor Action 888
••^'Genetic Studies on the Repressor Gene and theOperator Locus Lead to a Model for RepressorAction 888
Biochemical Investigations Verify the OperonHypothesis 889
An Activator Protein Is Discovered That AugmentsOperon Expression 890
Enzymes That Catalyze Amino Acid Biosynthesis AreRegulated at the Level of Transcription Initiation 891
The trp Operon Is Also Regulated after the InitiationPoint for Transcription 891
Genes for Ribosomes Are Coordinately Regulated 894Control of rRNA and tRNA Synthesis by the rel
Gene 894Translational Control of Ribosomal Protein
Synthesis 896Regulation of Gene Expression in Bacterial Viruses 896
A Metabolism Is Directed by Six RegulatoryProteins 897
The Dormant Prophage State of A Is Maintained by aPhage-Encoded Repressor 898 '
Events That Follow Infection of Escherichia coli byBacteriophage A Can Lead to Lysis orLysogeny 898
The N Protein Is an Antiterminator That Results inExtension of Early Transcripts 899
Another Antiterminator, the Q Protein, Is the Key toLate Transcription 900
Cm Protein Prevents Buildup of cl Protein during theLytic Cycle 900
Late Expression along the Lysogenic Pathway Requiresa Rapid Buildup of the ell Regulatory Protein 901
Interaction between DNA and DNA-Binding Proteins 902Recognizing Specific Regions in the DNA Duplex 902The Helix-Turn-Helix Is the Most Common Motif Found
in Prokaryotic Regulatory Proteins 903Helix-Turn-Helix Regulatory Proteins Are
Symmetrical 903DNA-Protein Cocrystals Reveal Gross Features of the
Complex 904From Cocrystal Studies, the Specific Contacts between
Base Pairs and Amino Acid Side Chains May BeDetermined 905
Some Regulatory Proteins Use the fi-Sheet Motif 909Involvement of Small Molecules in Regulatory Protein
Interaction 909RNA Can Act as a Repressor 909
C H A P T E R
Regulation of Gene Expression inEukaryotes 913Geoffrey Zubay
Gene^Regulation in Yeast: A Unicellular Eukaryote 915Galactose Metabolism Is Regulated by Specific Positive
dhd^ Negative Control Factors in Yeast 915The GAI$k:Protein Is Separated into Domains with
Different Functions 917
Contents xxi
Mating Type Is Determined by Transposable Elementsin Yeast 917
Gene Regulation in Multicellular Eukaryotes 919Nuclear Differentiation Starts in Early
Development 919Chromosome Structure Varies with Gene Activity 920
" Giant Chromosomes Permit Direct Visualization ofActive Genes 920
In Some Cases, Entire Chromosomes AreHeterochromatic 921
Biochemical Differences between Active and InactiveChromatin- 921
Histones May Play an Active Role inTranscription 922 f
Enhancers Are Promoter Elements That Operate overGreat Distances 923
DNA-Binding Proteins That Regulate Transcription inEukaryotes Are Often Asymmetrical 924
The Homeodomain 924Zinc Fingers 925Steroid Hormone Receptors Constitute a Special Class
of Zinc-Finger Regulatory Proteins 927 ~-Leucine Zipper 929Helix-Loop-Helix 929Transcription Activation Domains of Transcription
Factors ,929Alternative Modes of mRNA Splicing Present a Potent
Mechanism for-Posttranscriptional Regulation 930Gene Expression Is Also Regulated at the Levels of
Translation and Polypeptide Processing 930How Translation Controls Transcription in Eukaryotes 932Patterns of Regulation Associated with Developmental
Processes 932Early Development in Drosophila Leads to a Segmented
Structure Jha t Is Preserved to Adulthood 933Early Development in Drosophila Involves a Cascade
of Regulatory Events 933Three Types of Regulatory Genes Are Involved in Early
Segmentation Development in Drosophila 933Analysis of the Genes That Control the Early Events of
Drosophila Embryogenesis 934Cell-Cell Interaction Is Important in the Elaboration of
the Developmental Pattern in Parasegments 940Early Development in Drosophila and Vertebrates
Shows Striking Similarities 940
CHAPTER
Immunobiology 944Geoffrey Zubay
Overview of the Immune System 944The Humoral Response: B Cells and T Cells Working
Together 945Immunoglobulins Are Extremely Varied in Their
Specificities 945
Antibody Diversity Is Augmented by Unique Geneti:Mechanisms 947
Interaction of B Cells and T Cells Is Required for ",Antibody Formation 951 °
T Cell Action Is Frequently Augmented by the Secfy o
of Hormone-Like Proteins Called Interleukins ;:The Complement System Facilitates Removal of ,
Microorganisms and Antigen-Antibody iComplexes 954
The Cell-Mediated Response: A Separate Response by j °T Cells 955 \ •
Tolerance Prevents the Immune System from Attacl,Self-Antigens 955 '• ,
T Cells Recognize a Combination of Self andNonself 956 ';
MHC Molecules Account for Graft Rejection 956\v There Are Two Major Types of MHC Proteins: Cliu ;
and Class II 956 - J \T Cell Receptors Resemble Membrane-Bound •• •
Antibodies 956 "Additional Cell Adhesion Proteins Are Required to
Mediate the Immune Response 957 I °The Immune System in Action: A Broad Arsenal o); °
Weapons Enables the Immune System to Roust |i „Destroy Foreign Invaders 959 jj •
Immune Recognition Molecules Are EvolutionaryRelated 960 I
CHAPTER
Cancer and Carcinogenesis 963Geoffrey Zubay
Cancers Are Cells out of Control 964Environmental Factors Influence the Incidence of\
Cancers 964Cancerous Cells Are Almost Always Genetically '•
Abnormal 965Transformed Tissue Culture Cells Are Closely Reti
to Cancer Cells 966 IMany Tumors Arise by Mutational Events in Celln
Protooncogenes 967 IOncogenes Are Frequently Associated with Tumon
Causing Viruses 967 jThe Role of DNA Viral Genes in Transformation f
Reflects Their Role in the Permissive InfectioulCycle 968 \
Retroviral-Associated Oncogenes That Are Involved inj!Growth Regulation 969 j<
The src Gene Product 969 IThe sis Gene Product 969 IThe erbB Gene Product 970 ';
\ . x T h e ras Gene Product 970 ;'"The myc Gene Product 971 i|Thejun and fos Gene Products 972
• 2
xxii
The Transition from Protooncogene to Oncogene 973Tumor Suppressor Genes Are Genes Whose Presence Is
Needed to Block Transformation 973The Retinoblastoma Gene 973p53 Is the Most Common Gene Associated with Human
Cancers 973Understanding Cell Growth andCell Death Is Crucial to Our
Understanding of the Transition between Normal Cellsand Cancer Cells 975
How Close Are We to Understanding the Multistep ProcessThat Leads to Cancer? 975
Is There a Cure for Cancer? 976
C H A P T E R
The Human Immunodeficiency Virus (HIV)and Acquired Immunodeficiency Syndrome(AIDS) 979Geoffrey Zubay
Discovery and Incidence of AIDS 979
AIDS Is Associated with a Retrovirus 980Clinical Diagnosis of AIDS 981Is HIV Sufficient to Cause AIDS? 982HIV Belongs to the Cytopathic Subgroup of the
Retrovirus Family 982Molecular Biology of the HIV Virus 983
Tissue Specificity of HIV 983Course of the HIV Infection Leading to AIDS 983The HIV Genome 984 .The HIV Virus Life Cycle 984
Present Status and Future Prospects for the Prevention andTreatment of AIDS 986
Immunotherapy 987Drug Therapies 988Gene Therapy 988
Appendix A: Some Landmark Discoveries inBiochemistry A-l
Appendix B: Answers to Odd-Numbered ProblemsGlossary G-lCredits C-lIndex 1-1
A-5
Contents x xxiii
