Principles of biochemistry 4th ed h r horton, l moran, k g scrimgeour (pearson, 2006) 1

Horton, who received his Ph.D from the University of Missouri in 1962, is William Neal Reynolds Professor Emeritus and Alumni Distinguished Professor Emeritus in the Department of Bioche

Principles of

Biochemistry

Towson State University

Upper Saddle River, NJ 07458

PHOTO TO COME

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Principles of biochemistry / H Robert Horton [et al.].—4th ed

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About the cover: Complex III (ubiquinol:cytochrome c oxidoreductase) This

membrane-bound complex plays a key role in membrane-associated electron

transport and the generation of the proton gradient that eventually gives rise to new

ATP molecules Complex III catalyzes the Q-cycle reactions—one of the most

important pathways in biochemistry (See page 427.)

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iv

Science should be as simple

as possible, but not simpler.

—Albert Einstein

The Authors

H Robert Horton

Dr Horton, who received his Ph.D from the

University of Missouri in 1962, is William

Neal Reynolds Professor Emeritus and

Alumni Distinguished Professor Emeritus

in the Department of Biochemistry at North

Carolina State University, where he served

on the faculty for over 30 years Most of

Professor Horton’s research was in protein

and enzyme mechanisms

Laurence A Moran

After earning his Ph.D from Princeton

Uni-versity in 1974, Professor Moran spent four

years at the Université dè Geneve in

Switzerland He has been a member of the

Department of Biochemistry at the

Univer-sity of Toronto since 1978, specializing in

molecular biology and molecular evolution

His research findings on heat-shock genes

have been published in many scholarly

journals

K Gray Scrimgeour

Professor Scrimgeour received his

doctor-ate from the University of Washington in

1961 and has been a faculty member at the

University of Toronto since 1967 He is the

author of The Chemistry and Control of

En-zymatic Reactions (1977, Academic Press),

and his work on enzymatic systems has

been published in more than 50

profession-al journprofession-al articles during the past 40 years

From 1984–1992, he was editor of the

jour-nal Biochemistry and Cell Biology.

Marc D Perry

After earning his Ph.D from the

Universi-ty of Toronto in 1988, Dr Perry trained atthe University of Colorado, where he stud-ied sex determination in the nematode

C elegans In 1994 he returned to the

University of Toronto as a faculty member

in the department of Molecular and ical Genetics His research has focused ondevelopmental genetics, meiosis and bio-informatics In 2004 he joined the Heart

Med-& Stroke / Richard Lewar Centre ofExcellence in Cardiovascular Research inthe University of Toronto’s Faculty ofMedicine

J David Rawn

Professor Rawn, who received his Ph.Dfrom Ohio State University in 1971, hastaught and done research in the Department

of Chemistry at Towson State Universityfor the past 25 years He did not write chap-

ters for Principles of Biochemistry, but his textbook Biochemistry (1989, Neil Patter-

son) served as a source of information andideas concerning content and organization

New problems and solutions for the fourth edition were created by Drs Laurence A Moran,

University of Toronto and Elizabeth S Roberts-Kirchhoff, University of Detroit Mercy The

remaining problems were created by Drs Robert N Lindquist, San Francisco State

Univer-sity, Marc Perry and Diane M De Abreu of the University of Toronto

vii

PHOTO TO COME

THE BIOCHEMISTRY STUDENT COMPANION

by Allen J ScismCentral Missouri State University

No student should be without this helpful resource Contents include the following:

• carefully constructed drill problems for each chapter, including short-answer, choice, and challenge problems

multiple-• comprehensive, step-by-step solutions and explanations for all problems

• a remedial chapter that reviews the general and organic chemistry that students require forbiochemistry—topics are ingeniously presented in the context of a metabolic pathway

• tables of essential data

Please order through your college bookstore or call Prentice Hall at 1-800-947-7700.

The Biochemistry Student Companion

Structure and Function

3 Amino Acids and the Primary Structures of Proteins

4 Proteins: Three-Dimensional Structure and Function

10 Introduction to Metabolism

11 Glycolysis

12 Gluconeogenesis, The Pentose Phosphate Pathway,

and Glycogen Metabolism

13 The Citric Acid Cycle

14 Electron Transport and ATP Synthesis

20 DNA Replication, Repair, and Recombination

21 Transcription and RNA Processing

22 Protein Synthesis

23 Recombinant DNA Technology

Brief Contents

Preface xxv

PART ONE Introduction

1 Introduction to Biochemistry 1

1.1 Biochemistry Is a Modern Science 21.2 The Chemical Elements of Life 31.3 Many Important Macromolecules Are Polymers 5

A The Nucleus 18

B The Endoplasmic Reticulum and Golgi Apparatus 19

C Mitochondria and Chloroplasts 20

D Specialized Vesicles 21

E The Cytoskeleton 221.9 A Picture of the Living Cell 221.10 Biochemistry Is Multidisciplinary 24Appendix: The Special Terminology of Biochemistry 24Selected Readings 25

Contents

2 Water 26

2.1 The Water Molecule Is Polar 27

2.2 Hydrogen Bonding in Water 28

2.3 Water Is an Excellent Solvent 30

A Ionic and Polar Substances Dissolve in Water 30

B Cellular Concentrations and Diffusion 31

C Osmotic Pressure 312.4 Nonpolar Substances Are Insoluble in Water 32

PART TWO

Structure and Function

3 Amino Acids and the Primary Structures of Proteins 52

3.1 General Structure of Amino Acids 53

3.2 Structures of the 20 Common Amino Acids 55

Box 3.1 An Alternative Nomenclature 56

F Acidic R Groups and Their Amide Derivatives 60

G The Hydrophobicity of Amino Acid Side Chains 603.3 Other Amino Acids and Amino Acid Derivatives 61

3.4 Ionization of Amino Acids 62

3.5 Peptide Bonds Link Amino Acids in Proteins 66

3.6 Protein Purification Techniques 67

3.7 Analytical Techniques 69

3.8 Amino Acid Composition of Proteins 72

3.9 Determining the Sequence of Amino Acid Residues 73

3.10 Protein Sequencing Strategies 75

3.11 Comparisons of the Primary Structures of Proteins Reveal Evolutionary

Relationships 78

Contents xi

Summary 81Problems 81Selected Readings 83

4 Proteins: Three-Dimensional Structure and Function 84

4.1 There Are Four Levels of Protein Structure 864.2 Methods for Determining Protein Structure 874.3 The Conformation of the Peptide Group 90

4.6 Loops and Turns 974.7 Tertiary Structure of Proteins 98

C Van der Waals Interactions and Charge–Charge Interactions 112

D Protein Folding Is Assisted by Molecular Chaperones 1124.11 Collagen, a Fibrous Protein 115

4.12 Structures of Myoglobin and Hemoglobin 1164.13 Oxygen Binding to Myoglobin and Hemoglobin 118

A Oxygen Binds Reversibly to Heme 118

B Oxygen-Binding Curves of Myoglobin and Hemoglobin 119

C Hemoglobin Is an Allosteric Protein 1214.14 Antibodies Bind Specific Antigens 123Summary 125

Problems 125Selected Readings 127

A Derivation of the Michaelis–Menten Equation 137

B The Catalytic Constant 138

5.4 Kinetic Constants Indicate Enzyme Activity and Catalytic Proficiency 139

5.6 Kinetics of Multisubstrate Reactions 141

Box 5.1 Hyperbolas versus Straight Lines 141

a

5.10 Regulation of Enzyme Activity 148

A Phosphofructokinase Is an Allosteric Enzyme 149

B General Properties of Allosteric Enzymes 150

C Two Theories of Allosteric Regulation 152

D Regulation by Covalent Modification 1535.11 Multienzyme Complexes and Multifunctional Enzymes 154

Summary 154Problems 155Selected Readings 157

6.3 Chemical Modes of Enzymatic Catalysis 162

Box 6.1 Site-Directed Mutagenesis Modifies Enzymes 163

A Polar Amino Acid Residues in Active Sites 163

A The Proximity Effect 172

B Weak Binding of Substrates to Enzymes 172

A Zymogens Are Inactive Enzyme Precursors 182

B Substrate Specificity of Serine Proteases 183

C Serine Proteases Use Both the Chemical and the Binding Modes of Catalysis 184

Summary 188Problems 188Selected Readings 191

7 Coenzymes and Vitamins 192

7.1 Many Enzymes Require Inorganic Cations 1937.2 Coenzyme Classification 193

Box 7.1 Vitamin C: A Vitamin but Not a Coenzyme 1957.3 ATP and Other Nucleotide Cosubstrates 196

Box 7.2 NAD Binding to Dehydrogenases 199

7.7 Thiamine Pyrophosphate 2027.8 Pyridoxal Phosphate 203

Summary 218Problems 219Selected Readings 221

8.1 Most Monosaccharides Are Chiral Compounds 2238.2 Cyclization of Aldoses and Ketoses 226

8.3 Conformations of Monosaccharides 2298.4 Derivatives of Monosaccharides 231

A Structures of Disaccharides 234

B Reducing and Nonreducing Sugars 236

C Nucleosides and Other Glycosides 2368.6 Polysaccharides 237

A Starch and Glycogen 237

B Cellulose and Chitin 2398.7 Glycoconjugates 241

A Proteoglycans 241

Box 8.1 Nodulation Factors Are Lipo-oligosaccharides 243





9.1 Structural and Functional Diversity of Lipids 253

9.2 Fatty Acids 254

Box 9.1 Common Names of Fatty Acids 255

Box 9.2 Trans Fatty Acids and Margarine 2569.3 Triacylglycerols 258

9.4 Glycerophospholipids 259

9.5 Sphingolipids 262

9.7 Other Biologically Important Lipids 265

9.8 Biological Membranes Are Composed of Lipid Bilayers and Proteins 267

Box 9.3 Special Nonaqueous Techniques Must Be Used to Study Lipids 268

A Lipid Bilayers 269

B Fluid Mosaic Model of Biological Membranes 2709.9 Lipid Bilayers and Membranes Are Dynamic Structures 271

9.10 Three Classes of Membrane Proteins 274

Box 9.4 New Lipid Vesicles, or Liposomes 2759.11 Membrane Transport 278

A Thermodynamics of Membrane Transport 279

B Pores and Channels 280

C Passive Transport 281

D Active Transport 281

E Endocytosis and Exocytosis 283

Box 9.5 The Hot Spice of Chili Peppers 2849.12 Transduction of Extracellular Signals 284

A G Proteins Are Signal Transducers 285

B The Adenylyl Cyclase Signaling Pathway 287

C The Inositol–Phospholipid Signaling Pathway 288

Box 9.6 Bacterial Toxins and G Proteins 289

D Receptor Tyrosine Kinases 291Summary 292

Problems 292Selected Readings 294

Outer leaflet

B Metabolism Proceeds by Discrete Steps 300

C Metabolic Pathways Are Regulated 301

D Evolution of Metabolic Pathways 30310.3 Major Pathways in Cells 304

10.4 Compartmentation and Interorgan Metabolism 30610.5 Actual Gibbs Free Energy Change, Not Standard Free Energy Change, Determinesthe Spontaneity of Metabolic Reactions 308

10.6 The Free Energy of ATP 31010.7 The Metabolic Roles of ATP 313

A Phosphoryl-Group Transfer 314

B Production of ATP by Phosphoryl Group Transfer 315

C Nucleotidyl Group Transfer 31610.8 Thioesters Have High Free Energies of Hydrolysis 31710.9 Reduced Coenzymes Conserve Energy from Biological Oxidations 318

A Gibbs Free Energy Change Is Related to Reduction Potential 319

B Electron Transfer from NADH Provides Free Energy 322

Box 10.1 and NADH Differ in Their Ultraviolet Absorption Spectra 32210.10 Experimental Methods for Studying Metabolism 323

Summary 324Problems 324Selected Readings 326

11.1 The Enzymatic Reactions of Glycolysis 32811.2 The Ten Enzyme-Catalyzed Steps of Glycolysis 328

Box 11.1 A Brief History of the Glycolytic Pathway 332

Box 11.2 Formation of 2,3-Bisphosphoglycerate in Red Blood Cells 338

Box 11.3 Arsenate Poisoning 34011.3 The Fate of Pyruvate 340

A Metabolism of Pyruvate to Ethanol 341

B Reduction of Pyruvate to Lactate 34211.4 Free Energy Changes in Glycolysis 34311.5 Regulation of Glycolysis 344

A Regulation of Hexose Transporters 344

B Regulation of Hexokinase 346

Box 11.4 Glucose 6-Phosphate Has a Pivotal Metabolic Role in the Liver 346

C Regulation of Phosphofructokinase-1 347

D Regulation of Pyruvate Kinase 348

E The Pasteur Effect 35011.6 Other Sugars Can Enter Glycolysis 350

A Fructose Is Converted to Glyceraldehyde 3-Phosphate 350

B Galactose Is Converted to Glucose 1-Phosphate 351

C Mannose Is Converted to Fructose 6-Phosphate 35211.7 The Entner–Doudoroff Pathway in Bacteria 352Summary 354

Problems 354Selected Readings 355

α

SS

ββ

Insulin

Tyrosine-kinase

domains

Contents xvii

12 Gluconeogenesis, The Pentose Phosphate Pathway,

and Glycogen Metabolism 357

Box 12.1 Glucose Is Sometimes Converted to Sorbitol 36612.4 The Pentose Phosphate Pathway 366

A Oxidative Stage 368

B Nonoxidative Stage 368

Box 12.2 Glucose 6-phosphate Dehydrogenase Deficiency in Humans 369

C Interconversions Catalyzed by Transketolase and Transaldolase 37012.5 Glycogen Metabolism 371

A Glycogen Synthesis 371

B Glycogen Degradation 37212.6 Regulation of Glycogen Metabolism 374

A Hormones Regulate Glycogen Metabolism 375

B Reciprocal Regulation of Glycogen Phosphorylase and Glycogen Synthase 375

C Intracellular Regulation of Glycogen Metabolism Involves InterconvertibleEnzymes 376

Box 12.3 Glycogen Storage Diseases 37812.7 Maintenance of Glucose Levels in Mammals 379

Summary 381Problems 382Selected Readings 383

13 The Citric Acid Cycle 384

13.1 Conversion of Pyruvate to Acetyl CoA 385

13.2 The Citric Acid Cycle Oxidizes Acetyl CoA 391

13.3 The Citric Acid Cycle Enzymes 393

Box 13.1 Where Do the Electrons Come From? 394

Box 13.2 Three-point Attachment of Prochiral Substrates to Enzymes 397

Box 13.3 Converting One Enzyme into Another 40213.4 Reduced Coenzymes Can Fuel the Production of ATP 403

13.5 Regulation of the Citric Acid Cycle 404

13.6 The Citric Acid Cycle Isn’t Always a “Cycle” 406

13.7 The Glyoxylate Pathway 407

13.8 Evolution of the Citric Acid Cycle 410

Phosphorylation site

Allosteric binding site

Catalytic site

binding site

Glycogen-Summary 354Problems 354Selected Readings 355

14 Electron Transport and ATP Synthesis 415

14.1 Overview of Membrane-associated Electron Transport and ATP Synthesis 41614.2 The Mitochondrion 416

14.3 The Chemiosmotic Theory and the Protonmotive Force 418

A Historical Background: The Chemiosmotic Theory 418

B The Protonmotive Force 42014.4 Electron Transport 421

A Complexes I Through IV 421

B Cofactors in Electron Transport 42414.5 Complex I 424

14.6 Complex II 42514.7 Complex III 42714.8 Complex IV 42914.9 Complex V: ATP Synthase 432

Box 14.1 Proton Leaks and Heat Production 43514.10 Active Transport of ATP, ADP, and Pi Across the Mitochondrial Membrane 43514.11 The P/O Ratio 436

14.12 NADH Shuttle Mechanisms in Eukaryotes 436

Box 14.2 The High Cost of Living 43914.13 Other Terminal Electron Acceptors and Donors 43914.14 Superoxide Anions 440

Summary 441Problems 441Selected Readings 442

15.1 Light-Gathering Pigments 44515.2 Bacterial Photosystems 449

A Photosystem II 449

B Photosystem I 452

C Coupled Photosystems and Cytochrome bf 454

D Reduction Potentials and Gibbs Free Energy in Photosynthesis 458

E Photosynthesis Takes Place within Internal Membranes 45915.3 Plant Photosynthesis 460

A The Calvin Cycle 465

B Rubisco: Ribulose 1,5-bisphosphate Carboxylase–oxygenase 465

C Oxygenation of Ribulose 1,5-Bisphosphate 469

D Calvin Cycle: Reduction and Regeneration Stages 470

Box 15.2 Building a Better Rubisco 470

Heme b

QH2

Membrane OUTSIDE

15.5 Sucrose and Starch Metabolism in Plants 471

15.6 Additional Carbon-Fixation Pathways 473

Box 15.3 Gregor Mendel’s Wrinkled Peas 473

A The Pathway 474

B Crassulacean Acid Metabolism (CAM) 474

C Carbon Fixation in Bacteria 476Summary 477

Problems 477Selected Readings 478

16.1 Fatty Acid Synthesis 480

A Synthesis of Malonyl ACP and Acetyl ACP 480

B The Initiation Reaction of Fatty Acid Synthesis 481

C The Elongation Reactions of Fatty Acid Synthesis 482

D Activation of Fatty Acids 483

E Fatty Acid Extension and Desaturation 48416.2 Synthesis of Triacylglycerols and Glycerophospholipids 485

16.3 Synthesis of Eicosanoids 488

Box 16.1 The Search for a Replacement for Aspirin 49016.4 Synthesis of Ether Lipids 490

16.5 Synthesis of Sphingolipids 491

Box 16.2 Lysosomal Storage Diseases 493

Box 16.3 Regulating Cholesterol Levels 49416.6 Synthesis of Cholesterol 495

A Stage 1: Acetyl CoA to Isopentenyl Diphosphate 495

B Stage 2: Isopentenyl Diphosphate to Squalene 496

C Stage 3: Squalene to Cholesterol 496

D Other Products of Isoprenoid Metabolism 49616.7 Fatty Acid Oxidation 498

C Transport of Fatty Acyl CoA into Mitochondria 501

Box 16.4 A Trifunctional Enzyme for 502

D ATP Generation from Fatty Acid Oxidation 502

16.8 Eukaryotic Lipids Are Made at a Variety of Sites 506

16.9 Lipid Metabolism Is Regulated by Hormones in Mammals 507

16.10 Absorption and Mobilization of Fuel Lipids in Mammals 509

A Absorption of Dietary Lipids 509

B Lipoproteins 510

Box 16.5 Lipoprotein Lipase and Coronary Heart Disease 513

C Serum Albumin 51316.11 Ketone Bodies Are Fuel Molecules 513

A Ketone Bodies Are Synthesized in the Liver 514

B Ketone Bodies Are Oxidized in Mitochondria 515

Box 16.6 Altered Carbohydrate and Lipid Metabolism in Diabetes 516Summary 517

b-Oxidation

b-Oxidation

b-Oxidationb-Oxidation

C4

Contents xix

Problems 517Selected Readings 519

17.1 The Nitrogen Cycle and Nitrogen Fixation 52117.2 Assimilation of Ammonia 523

A Ammonia Is Incorporated into Glutamate and Glutamine 524

B Transamination Reactions 52417.3 Synthesis of Amino Acids 526

A Aspartate and Asparagine 526

Box 17.1 Childhood Acute Lymphoblastic Leukemia Can Be Treated with

Asparaginase 526

B Lysine, Methionine, and Threonine 527

C Alanine, Valine, Leucine, and Isoleucine 528

D Glutamate, Glutamine, Arginine, and Proline 529

E Serine, Glycine, and Cysteine 530

F Phenylalanine, Tyrosine, and Tryptophan 531

Box 17.2 Genetically Modified Food

Box 17.3 Essential and Nonessential Amino Acids in Animals

G Histidine 53517.4 Amino Acids as Metabolic Precursors 536

A Products Derived from Glutamate, Glutamine, and Aspartate 536

B Products Derived from Serine and Glycine 536

C Synthesis of Nitric Oxide from Arginine 53617.5 Protein Turnover 538

Box 17.4 Apoptosis—Programmed Cell Death 53817.6 Amino Acid Catabolism 539

A Alanine, Asparagine, Aspartate, Glutamate, and Glutamine 541

B Arginine, Histidine, and Proline 541

C Glycine and Serine 542

D Threonine 543

E The Branched-Chain Amino Acids 543

F Methionine 545

G Cysteine 546

H Phenylalanine, Tryptophan, and Tyrosine 546

Box 17.5 Phenylketonuria, a Defect in Tyrosine Formation 546

I Lysine 548

Box 17.6 Diseases of Amino Acid Metabolism 54817.7 The Urea Cycle Converts Ammonia into Urea 549

A Synthesis of Carbamoyl Phosphate 549

B The Reactions of the Urea Cycle 549

C Ancillary Reactions of the Urea Cycle 550

Box 17.7 The Liver Is Organized for Removing Toxic Ammonia 55117.8 Renal Glutamine Metabolism Produces Bicarbonate 553

Summary 554Problems 555Selected Readings 556

Contents xxi

18 Nucleotide Metabolism 557

18.1 Synthesis of Purine Nucleotides 558

18.2 Other Purine Nucleotides Are Synthesized from IMP 561

Box 18.1 Common Names of the Bases 56118.3 Synthesis of Pyrimidine Nucleotides 563

A The Pathway for Pyrimidine Synthesis 564

Box 18.2 How Some Enzymes Transfer Ammonia from Glutamine 565

B Regulation of Pyrimidine Synthesis 56618.4 CTP Is Synthesized from UMP 568

18.5 Reduction of Ribonucleotides to Deoxyribonucleotides 569

18.6 Methylation of dUMP Produces dTMP 570

Box 18.3 Free Radicals in the Reduction of Ribonucleotides 570

Box 18.4 Cancer Drugs Inhibit dTTP Synthesis 57218.7 Salvage of Purines and Pyrimidines 573

PART FOUR

Biological Information Flow

19 Nucleic Acids 583

19.1 Nucleotides Are the Building Blocks of Nucleic Acids 584

A Ribose and Deoxyribose 584

B Purines and Pyrimidines 584

C Nucleosides 586

D Nucleotides 58719.2 DNA Is Double-Stranded 590

A Nucleotides Are Joined by Phosphodiester Linkages 590

B Two Antiparallel Strands Form a Double Helix 592

C Weak Forces Stabilize the Double Helix 595

D Conformations of Double-Stranded DNA 59719.3 DNA Can Be Supercoiled 597

19.4 Cells Contain Several Kinds of RNA 599

19.5 DNA Is Packaged in Chromatin in Eukaryotic Cells 599

A Nucleosomes 600

Box 19.1 Histones Can Be Acetylated and Deacetylated 601

B Higher Levels of Chromatin Structure 603

C Bacterial DNA Packaging 60419.6 Nucleases and Hydrolysis of Nucleic Acids 605

A Alkaline Hydrolysis of RNA 605

B Ribonuclease-Catalyzed Hydrolysis of RNA 605

C Restriction Endonucleases 608

D EcoRI Binds Tightly to DNA 610

3¿ – 5¿

19.7 Uses of Restriction Endonucleases 610Summary 612

Problems 612Selected Readings 613

20 DNA Replication, Repair, and Recombination 615

20.1 Chromosomal DNA Replication Is Bidirectional 61620.2 DNA Polymerase 618

A Chain Elongation Is a Nucleotidyl-Group-Transfer Reaction 619

B DNA Polymerase III Remains Bound to the Replication Fork 621

C Proofreading Corrects Polymerization Errors 62120.3 DNA Polymerase Synthesizes Two Strands Simultaneously 622

A Lagging-Strand Synthesis Is Discontinuous 623

B Each Okazaki Fragment Begins with an RNA Primer 623

C Okazaki Fragments Are Joined by the Action of DNA Polymerase I and DNA Ligase 624

20.4 Model of the Replisome 62620.5 Initiation and Termination of DNA Replication 62920.6 DNA Replication in Eukaryotes 630

Box 20.1 Sequencing DNA Using Dideoxynucleotides 63220.7 Repair of Damaged DNA 634

A Repair after Photodimerization: An Example of Direct Repair 635

B Excision Repair 63520.8 Homologous Recombination 639

A The Holliday Model of General Recombination 639

B Recombination in E coli 640

C Recombination Can Be a Form of Repair 641

Box 20.2 Molecular Links Between DNA Repair and Breast Cancer 643Summary 644

Problems 645Selected Readings 646

21 Transcription and RNA Processing 647

21.1 Types of RNA 64821.2 RNA Polymerase 649

A RNA Polymerase Is an Oligomeric Protein 649

B The Chain Elongation Reaction 65021.3 Transcription Initiation 652

A Genes Have a Orientation 652

B The Transcription Complex Assembles at a Promoter 652

C The Subunit Recognizes the Promoter 655

D RNA Polymerase Changes Conformation 65521.4 Transcription Termination 656

21.5 Transcription in Eukaryotes 659

A Eukaryotic RNA Polymerases 659

B Eukaryotic Transcription Factors 662

C The Role of Chromatin in Eukaryotic Transcription 66321.6 Transcription of Genes Is Regulated 663

s

5¿ : 3¿

Contents xxiii

21.7 The lac Operon, an Example of Negative and Positive Regulation 665

A lac Repressor Blocks Transcription 665

B The Structure of lac Repressor 667

C cAMP Regulatory Protein Activates Transcription 66821.8 Posttranscriptional Modification of RNA 670

A Transfer RNA Processing 671

B Ribosomal RNA Processing 67221.9 Eukaryotic mRNA Processing 674

A Eukaryotic mRNA Molecules Have Modified Ends 674

B Some Eukaryotic mRNA Precursors Are Spliced 677Summary 680

Problems 680Selected Readings 682

22 Protein Synthesis 683

22.1 The Genetic Code 683

22.2 Transfer RNA 686

A The Three-Dimensional Structure of tRNA 686

B tRNA Anticodons Base-Pair with mRNA Codons 68822.3 Aminoacyl-tRNA Synthetases 688

A The Aminoacyl-tRNA Synthetase Reaction 689

B Specificity of Aminoacyl-tRNA Synthetases 689

C Proofreading Activity of Aminoacyl-tRNA Synthetases 691

A Ribosomes Are Composed of Both Ribosomal RNA and Protein 693

B Ribosomes Contain Two Aminoacyl-tRNA Binding Sites 69522.5 Initiation of Translation 695

A Initiator tRNA 695

B Initiation Complexes Assemble Only at Initiation Codons 695

C Initiation Factors Help Form the Initiation Complex 696

D Translation Initiation in Eukaryotes 69722.6 Chain Elongation Is a Three-Step Microcycle 697

A Elongation Factors Dock an Aminoacyl-tRNA in the A Site 699

B Peptidyl Transferase Catalyzes Peptide Bond Formation 700

C Translocation Moves the Ribosome by One Codon 70122.7 Termination of Translation 705

22.8 Protein Synthesis Is Energetically Expensive 705

22.9 Regulation of Protein Synthesis 705

A Ribosomal Protein Synthesis Is Coupled to Ribosome Assembly

in E coli 706

Box 22.1 Some Antibiotics Inhibit Protein Synthesis 707

B Globin Synthesis Depends on Heme Availability 707

C The E coli trp Operon Is Regulated by Repression and Attenuation 70822.10 Posttranslational Processing 712

A The Signal Hypothesis 712

B Glycosylation of Proteins 716Summary 716

Problems 717Selected Readings 718

21S particle

Complete 30S subunit

23 Recombinant DNA Technology 719

23.1 Making Recombinant DNA 71923.2 Cloning Vectors 721

23.7 Chromosome Walking 73323.8 Expression of Proteins Using Recombinant DNA Technology 734

A Prokaryotic Expression Vectors 734

B Expression of Proteins in Eukaryotes 73423.9 Applications of Recombinant DNA Technology 735

A Genetic Engineering of Plants 737

B Genetic Engineering in Prokaryotes 73723.10 Applications to Human Diseases 73923.11 The Polymerase Chain Reaction Amplifies Selected DNA Sequences 741

Box 23.2 Medical Uses of PCR 74123.12 Site-Directed Mutagenesis of Cloned DNA 743Summary 744

Problems 745Selected Readings 747

Solutions 749 Illustration Credits 809 Glossary 811

Index 827

b – Galactosidasel

To the Student

Welcome to biochemistry—the study of the of life at the molecular level As you

venture into this exciting and dynamic discipline, you’ll discover many new and

wonderful things You’ll learn how some enzymes can catalyze chemical reactions

at speeds close to theoretical limits—reactions that would otherwise occur only at

imperceptibly low rates You’ll learn about the forces that maintain biomolecular

structure and how even some of the weakest of those forces make life possible.

You’ll also learn how biochemistry has thousands of applications in day-to-day

life—in medicine, drug design, nutrition, forensic science, agriculture, and

manu-facturing In short, you’ll begin a journey of discovery about how biochemistry

makes life both possible and better.

Before we begin, we would like to offer a few words of advice:

Don’t just memorize facts; instead, understand principles

In this book, we have tried to identify the most important principles of

biochem-istry Every year, a million or so research papers are published Half of them

de-scribe the results of research in some area of biochemistry Because the knowledge

base of biochemistry is continuously expanding, we must grasp the underlying

themes of this science in order to understand it This textbook is designed to

ex-pand on the foundation you have acquired in your chemistry and biology courses

and to provide you with a biochemical framework that will allow you to understand

new phenomena as you meet them As you progress in your studies, you will

en-counter many examples that flesh out the framework we describe in this book.

These individual facts are useful in illuminating the basic principles.

Be prepared to learn a new vocabulary

An understanding of biochemical facts requires that you learn a biochemical

vo-cabulary This vocabulary includes the chemical structures of a number of key

mol-ecules These molecules are grouped into families based on their structures and

functions You will also learn how to distinguish among members of each family

and how small molecules combine to form macromolecules such as proteins and

nucleic acids As with any newly studied discipline, the more familiar you are with

the vocabulary the more easily you can learn and appreciate the scientific literature.

Test your understanding

True mastery of biochemistry lies with learning how to apply your knowledge and

how to solve problems Each chapter concludes with a set of carefully crafted

prob-lems that test your understanding of core principles Many of these probprob-lems are

mini case studies that present the problem within the context of a real biochemical

puzzle.

Preface

PHOTO TO COME

For more practice, including solving more traditional review exercises in

bio-chemistry, we are pleased to refer you to The Biochemistry Student Companion by

Allen Scism (see page viii) This book presents a variety of supplementary

ques-tions that you may find helpful You will also find additional problems on the

Learn to visualize in 3-D

Biochemicals are three-dimensional objects Understanding what happens in a chemical reaction at the molecular level requires that you be able to “see” what happens in three dimensions We present the structures of simple molecules in sev- eral different ways in order to illustrate their three-dimensional conformation The structures of proteins and protein complexes are also drawn as three-dimensional objects Throughout this book you will find many excellent drawings by our expe- rienced team of artists In addition, on the website you will find many animations and interactive molecular models that you can manipulate in real-time on a com- puter We strongly suggest you look at these movies and do the exercises that ac- company them as well as participate in the molecular visualization tutorials.

bio-Feedback

Finally, please let us know of any errors or omissions you encounter as you use this text Tell us what you would like to see in the next edition Many of the changes in this fourth edition began with suggestions, and criticisms, from students like you With your help, we will continue to evolve this work into an even more useful tool Our e-mail addresses are at the end of this Preface Good luck, and enjoy!

To the Instructor

We welcome all our loyal users and those instructors who are, for the first time, teaching courses in biochemistry This textbook is intended for students in one- semester courses but it is becoming more and more popular in two-semester courses We hope you will find this the ideal book for your course If you have any questions or comments for us, please get in touch with us by e-mail.

A book for your students

We have organized this book as we would organize our own courses We have cluded all the topics that are usually covered in introductory biochemistry courses although we are well aware of the fact that no single course covers every topic and every chapter At some point, students cease to be intimidated by the need to skip around in a book In our experience, that transition lies somewhere after they have begun a course like yours.

in-We have tried to make the text as modular as possible Each large topic resides

in its own section Reaction mechanisms are often separated from the main thread

of the text and can be passed over by those who prefer not to cover this level of detail The text is extensively cross-referenced to make it easier for you to reorga- nize the chapters and for students to see the interrelationships among various topics and to drill down to deeper levels of understanding.

We built the book explicitly for the beginning student taking a first course in this burgeoning subject Parts One and Two lay a solid foundation of chemical knowledge that will help students understand, rather than merely memorize, the dynamics of metabolic and genetic processes These sections assume that students have taken prerequisite courses in general and organic chemistry and have acquired

a rudimentary knowledge of the organic chemistry of carboxylic acids, amines,

Preface xxvii

alcohols, and aldehydes Even so, key functional groups and chemical properties of

each type of biomolecule are carefully explained as their structures and functions

are presented.

A focus on principles

There are, in essence, two kinds of biochemistry textbooks: those for reference and

those for teaching It is difficult for one book to be both as it is those same thickets

of detail sought by the professional that ensnare the struggling novice on his or her

first trip through the bush This text is unapologetically a text for teaching It has

been designed to foster student understanding and is not an encyclopedia of

bio-chemistry This book focuses unwaveringly on teaching basic principles, each

prin-ciple supported by carefully chosen examples.

A focus on chemistry with rigorous biology

When we first wrote this text, we decided to take the time to explain in chemical

terms the principles we underscore To that end, we offer detailed chemical

expla-nations of most of the chemistry herein, including mechanisms (which tell students

how and why things happen).

While we emphasize chemistry, we also stress the bio in biochemistry We

point out that biochemical systems evolve and that the reactions that occur in some

species are variations on a larger theme In this edition, we increase our emphasis

on the similarities of prokaryotic and eukaryotic systems, while we continue to

avoid making generalizations about all organisms based on reactions that occur in

a few.

We are proud of the fact that this is the most scientifically accurate istry textbook We have gone to great lengths to ensure that our facts are correct

biochem-and our explanations of basic concepts reflect the modern consensus among active

researchers Our success is due, in large part, to the dedication of our many

review-ers and editors.

A focus on the visual

Biochemistry is a three-dimensional science Our inclusion of the latest

computer-generated images is intended to clarify the shape and function of molecules and to

leave students with an appreciation for the relationship between the structure and

function Most of the protein images in this edition are new; they have been

skill-fully prepared by Jonathan Parrish of the University of Alberta.

For those students with access to a computer, we offer a number of other portunities We have included Protein Data Bank (PDB) reference numbers for the

op-coordinates from which all protein images were derived This allows students to

explore the structures further on their own In addition, we have a gallery of

pre-pared PDB files that students can view using Chime or any other molecular viewer;

these are posted on the text’s Companion Website (http://www.prenhall.

com/horton) as are animations of key dynamic processes as well as visualization

tutorials using Chime Finally, we offer instructors a CD that contains the gallery of

animations, all the PDB files, as well as most of the art from the textbook in jpg

format This makes the preparation of in-class electronic presentations (e.g., using

PowerPoint) much easier Please see the listing of supplements on page xxxii.

The organization

We strive to present the story of biochemistry in a clear, coherent, well-integrated

manner, building at each stage the background needed for the next stage This book

is arranged in four parts, each building on those that come before.

Part One forms an introduction to all that follows In Chapter 1 we have added sections on thermodynamics and reaction kinetics in response to suggestions from

your colleagues These short descriptions will help students who need to review

basic chemical principles Chapter 1 also includes a brief survey of cellular ture and the four classes of macromolecules—proteins, carbohydrates, lipids, and nucleic acids In this chapter, and throughout the book, we sustain a consistent pat- tern of presentation, from basic chemistry to biochemical function We first show the chemistry of monomeric units and then explore the properties and functions

struc-of the biopolymers and aggregates formed from those monomers—amino acids to proteins, monosaccharides to polysaccharides and glycoconjugates, lipids to mem- branes, and nucleotides to nucleic acids.

Part Two includes three chapters on enzyme properties, enzyme mechanisms, and coenzymes (Chapters 5, 6, and 7) We encourage a firm grasp of these critical subjects since they are required for later appreciation of the role of enzymes and coenzymes in metabolic pathways Chapter 8 covers carbohydrates and their derivatives We have added new information on the ABO blood groups in the sec- tion on glycoproteins Lipids and membranes are described in Chapter 9 and this

is where we introduce students to the exciting field of signal transduction Our description of membrane transport and membrane potentials has been improved

in order to better prepare students for chapters on electron transport and proton gradients.

Part Three features nine chapters that focus on metabolic pathways In ter 10, we introduce the intricate molecular symphonies of metabolism by consid- ering how pathways are energized, interrelated, and regulated In this edition we have added new material on how metabolic pathways evolve—a theme that we re- turn to in subsequent chapters Chapter 11 describes glycolysis in detail At this stage, we establish a format that recurs in subsequent metabolic chapters—first describing the basic pathway in chemical and enzymatic terms, then demonstrating the bioenergetic requirements and sources of energy, and concluding with an account of regulatory mechanisms Note that the early introduction of signal trans- duction (Chapter 9) allows integration of regulation into each chapter of metabo- lism and allows discussion of flux through reciprocal pathways.

Chap-Chapter 12 has been completely reorganized We now place a greater emphasis

on gluconeogenesis and we explain how the pathways of glycolysis and genesis are related Most biochemistry teachers are spending more time on gluco- neogenesis since it is the fundamental pathway in most species In the following chapters on lipid metabolism (Chapter 16), amino acid metabolism (Chapter 17), and nucleotide metabolism (Chapter 18), we describe the biosynthesis pathways be- fore showing the degradation pathways Again, this is consistent with our focus on the basic principles of biochemistry that apply to most species and it reflects a new trend in teaching biochemistry Whenever possible we explain where human bio- chemical pathways differ from the ones seen in other species.

gluconeo-We hope you enjoy the new look of Chapter 13 (Citric Acid Cycle) and ter 14 (Electron Transport and ATP Synthesis) We have put far more emphasis on the evolution of these pathways and the relationship between prokaryotic and eu- karyotic versions We have also incorporated new structures of membrane com- plexes as part of a strategy to relate structure and function in a more meaningful way than was possible with previous editions Part Three also includes a com- pletely revised chapter on photosynthesis (Chapter 15) We have taken advantage

Chap-of the tremendous amount Chap-of new information on photosynthesis to describe how simple bacterial systems evolved into the more complex plant systems We have also paid more attention to integrating the principles of photosynthesis and the material on chemiosmotic theory introduced in the preceding chapter on electron transport.

Part Four concludes the book with five chapters on the flow of biological formation Our main emphasis throughout this series of chapters is on the basic processes that govern pathways of information flow We take pains to incorporate within our story of genes and gene expression the principles of biochemistry taught

in-in precedin-ing chapters The fin-inal chapter (Chapter 23) is a substantial and porary presentation of techniques used in recombinant DNA technology.

contem-Preface xxix

New features in this edition

We are grateful for all the input we received on the first three editions of this text.

In addition to the changes noted above, you’ll notice the following improvements

in this fourth edition:

• expanded descriptions of oxidation-reduction reactions in several chapters,

in-cluding more explanation of where electrons come from.

• additional problems at the end of every chapter, with detailed solutions to all

problems in an appendix at the end of the book.

• more sample solutions to problems.

• more extensive marginal cross-referencing to enhance the connection of

con-cepts and the integration of material, to permit instructors to cover the

materi-al in a different order, and to improve the efficiency of students’ studying.

• the addition of special topic, clinical application, and deeper-look boxes to

most chapters.

• extra boxes on the origins of biochemical names and terms in order to make

the vocabulary more friendly and interesting.

• expansion of the complete glossary of biochemical terms at the end of the

book.

• update of all Selected Readings at the end of each chapter.

Since publication of the third edition, major advances have been made in a number of areas For this edition, we made changes to each section of the text to re-

flect these advances For example, we added many new protein structures, each

with Protein Data Bank cross-references; expanded the discussion of protein

struc-ture (with more examples); expanded the discussion of known multifunctional

en-zymes; increased the emphasis on molecular evolution; enhanced the discussion of

the mechanism of energy conservation as ATP; incorporated the results of the

vari-ous genome initiatives; and added new information on transcription and

transla-tion, including new information on the structures of the ribosome and RNA

polymerase.

We have created a student learning package that includes the following:

• The Biochemistry Student Companion

• Companion Website

The instructor’s packages consist of

• Instructor Resource Center on CD/DVD

• test item file,

• Transparency Set of 150 full-color acetates and masters,

• OneKey solution to course management.

Acknowledgments

We are grateful to our many talented and thoughtful reviewers who have helped

shape this book.

Reviewers who helped in the Fourth Edition:

Accuracy Reviewers David Watt, University of Kentucky Neil Haave, University of Alberta

Content Reviewers Consuelo Alvarez, Longwood University Marilee Benore Parsons, University of Michigan Albert M Bobst, University of Cincinnati Gary J Blomquist, University of Nevada, Reno Kelly Drew, University of Alaska, Fairbanks Andrew Feig, Indiana University

Giovanni Gadda, Georgia State University Donna L Gosnell, Valdosta State University Charles Hardin, North Carolina State University Jane E Hobson, Kwantlen University College Ramji L Khandelwal, University of Saskatchewan Scott Lefler, Arizona State

Kathleen Nolta, University of Michigan Jeffrey Schineller, Humboldt State University Richard Shingles, Johns Hopkins University Michael A Sypes, Pennsylvania State University Martin T Tuck, Ohio University

Julio F Turrens, University of South Alabama David Watt, University of Kentucky

James Zimmerman, Clemson University Reviewers who have helped with previous editions:

Lawrence Aaronson, Utica College of Syracuse; Stephen L Bearne, University of North Carolina at Chapel Hill; Robert Bergen, University of Central Arkansas; Gary J Blomquist, University of Nevada–Reno; Robert I Bolla, Saint Louis Uni- versity; Lori Bolyard, University of Evansville; John Brosnan, Memorial Univer- sity of Newfoundland; Ronald Callahan, New York University; Martin F Chaplin, South Bank University; William Coleman, University of Hartford; Harold Cook, Dalhousie University; Gary W Daughdrill, University of Idaho; Ruthellen M Daw- ley, University of Evansville; John Durham, West Virginia School of Medicine; Yves Engelborghs, University of Leuven; Edward Funkhouser, Texas A&M Uni- versity; Milton Gordon, University of Washington; Kenneth, E Guyer, Marshall University; Robert Harris, Indiana University–School of Medicine; Jan Hoek, Thomas Jefferson University; J Kenneth Hoober, Arizona State University; Cristi Hunnes, Rocky Mountain College; Mahendra K Jain, University of Delaware; Thomas D Kim, Youngstown State University; David Koetje, SUNY–Fredonia; James A Knopp, North Carolina State University; Susan Lees-Miller, Roger A Lewis, University of Nevada at Reno; University of Calgary; Robert N Lindquist, San Francisco State University; Richard Lomneth, University of Nebraska at Omaha; Ray Lutgring, University of Evansville; George Marzluf, Ohio State Uni- versity; Barbara Olson, University of Calgary; Thomas Prasthofer, Albany College

of Pharmacy; Thomas Reilly, California State University–Dominguez Hills; Carl Rhodes, University of Illinois–Urbana-Champagne; Gale Rhodes, University of Southern Maine; Duane L Rohlfing, University of South Carolina; Douglas Rus- sell, Dalhousie University; Aziz Sancar, University of North Carolina-School of Medicine; Larry Scheve, California State University—Hayward; Allen Scism, Central Missouri State University; Steven Seifried, University of Hawaii at Manoa; Thomas Sherman, University of Pittsburgh; Timothy A Sherwood, Arkansas Tech University; Dean Sherry, University of Texas at Dallas; David Skalnik, Indiana University School of Medicine; Anthony F Sky, Lawrence Technological Univer- sity; Gary D Small, University of South Dakota; Ronald L Somerville, Purdue University; Ralph Stephani, St John’s University; Laurence Tate, University of South Alabama; William Thompson, University of Toronto; Richard W Topham, University of Richmond; Arrel Towes, University of South Carolina; Julio F Turrens, University of South Alabama; Jack Y Vanderhoek, Charles Waechter,

University of Kentucky; Jubran M Wakim, Middle Tennessee State University;

George Washington University; William Wolodko, University of Alberta; David

Yang, Georgetown University

We would also like to thank our colleagues who have previously contributed material for particular chapters and whose careful work still inhabits this book:

Roy Baker, University of Toronto Roger W Brownsey, University of British Columbia Willy Kalt, Agriculture Canada

Robert K Murray, University of Toronto Frances Sharom, University of Guelph Malcolm Watford, Rutgers, The State University of New Jersey Putting this book together was a collaborative effort, and we would like to thank various members of the team who have helped give this project life: Jonathan

Parrish, Jay McElroy, Lisa Shoemaker, and the artists of Prentice Hall; Jennifer

Hart, editorial assistant, Crissy Dudonis, Project Editor in charge of supplements,

Patrick Shriner, Media Editor, Andrew Gilfillan, Marketing Manager; and a special

thanks to Marty Sopher, our Production Editor, whose organizational skills made

this book happen We would also like to thank Gary Carlson, our Executive Editor

at Prentice Hall.

Finally, we close with an invitation for feedback Despite our best efforts (and

a terrific track record in the previous editions), there are bound to be mistakes in a

work this size We are committed to making this the best biochemistry text

avail-able; please know that all comments are welcome.

Guide to the Features of the Text

Most students who purchase this book aim for mastery of the material They also want a good grade

in the course Here’s some advice to help you attain these goals.

• Attend lectures and take notes.

• Keep up with assignments.

• Read this textbook as assigned by your instructor.

• Practice your skills by solving problems—many are like the ones on your exams.

348 CHAPTER 11 ■ Glycolysis

OH

HO H

H 2 3 4 5

CH 2 OH

OH O

H

H 2 O b- D -Fructose 6-phosphate

H 2 3 4 5

CH 2 OH O 2

6

⌷ 3 POCH 2

Figure 11.16

Interconversion of -D-fructose 6-phosphate

and -D-fructose 2,6-bisphosphate b

Citrate, an intermediate of the citric acid cycle, is another physiologically portant inhibitor of PFK-1 An elevated concentration of citrate indicates that the citric acid cycle is blocked and further production of pyruvate would be pointless.

im-The regulatory effect of citrate on PFK-1 is an example of feedback inhibition that regulates the supply of pyruvate to the citric acid cycle.

The activity of PFK-1 is also regulated by the intracellular pH For example, during heavy exercise, anaerobic glycolysis produces lactic acid in muscle cells

If the lactic acid is not removed fast enough by the blood, the pH decreases and PFK-1 is inhibited by the excess hydrogen ions.

Fructose 2,6-bisphosphate (Figure 11.16) is a potent activator of PFK-1,

effec-tive in the micromolar range This compound is present in mammals, fungi, and plants, but not prokaryotes.

Fructose 2,6-bisphosphate is formed from fructose 6-phosphate by the action

of the enzyme phosphofructokinase-2 (PFK-2, or fructose 6-phosphate, 2-kinase).

PFK-2 is stimulated by inorganic phosphate and inhibited by citrate Surprisingly,

in mammalian liver, a different active site on the same protein catalyzes the

hy-drolytic dephosphorylation of fructose 2,6-bisphosphate, re-forming fructose 6-phosphate This activity of the enzyme is called fructose 2,6-bisphosphatase The

phosphate.

In the liver, the activity of PFK-2 is linked to the action of glucagon, a mone produced by the pancreas in response to low blood glucose levels An eleva- tion of the glucagon concentration in the blood triggers the adenylyl cyclase signaling pathway in liver cells, culminating in the phosphorylation of a serine residue in PFK-2 (Figure 11.17) Phosphorylation inactivates the kinase activity of the bifunctional enzyme and activates its phosphatase activity Under these condi-

hor-tions, the level of fructose 2,6-bisphosphate falls, PFK-1 becomes less active, and

glycolysis is depressed Under conditions in which glucose is rapidly metabolized,

more fructose 2,6-bisphosphate is formed since fructose 6-phosphate is both a strate of PFK-2 and a potent inhibitor of fructose 2,6-bisphosphatase A phospho-

sub-protein phosphatase catalyzes the dephosphorylation of PFK-2 Thus, in liver cells, control of glycolysis by glucagon and glucose is accomplished through control of the bifunctional enzyme whose activity establishes the steady-state concentration

of fructose 2,6-bisphosphate.

D Regulation of Pyruvate Kinase

The third site of allosteric regulation of glycolysis is the reaction catalyzed by pyruvate kinase Four different isozymes of pyruvate kinase are present in mam- moidal curve when initial velocity is plotted against phosphoenolpyruvate concentration (Figure 11.18a) These enzymes are allosterically activated by fruc-

tose 1,6-bisphosphate (Figure 11.19, on page 350) and inhibited by ATP In the sence of fructose 1,6-bisphosphate, physiological concentrations of ATP almost completely inhibit the isolated enzyme The presence of fructose 1,6-bisphos- phate—probably the most important modulator in vivo—shifts the curve to the left.

ab-With sufficient fructose 1,6-bisphosphate, the curve becomes hyperbolic Figure

11.18a shows that enzyme activity is greater in the presence of the allosteric

acti-vator for a range of substrate concentrations Recall that fructose 1,6-bisphosphate the activity of PFK-1 increases Since fructose 1,6-bisphosphate activates pyruvate

▲ Complete Explanations of the Chemistry

There are thousands of metabolic reactions in humans You might try to memorize them all, but eventually you’ll run out of memory What’s more, memorization won’t help you if you encounter something you haven’t seen before.

In this book, we show you some of the basic mechanisms of enzyme-catalyzed reactions—an extension

of what you learned in organic chemistry If you understand the mechanism, you’ll understand the chemistry You’ll have less to memorize, and you’ll retain the information more effectively.

xxxii

Margin Notes

There is a great deal of detail in biochemistry, but we want you to see both the forest and the trees When

we need to cross-reference something discussed earlier in the book, or something that we will come back to later, we put it in the margin.

Backward references offer a review

of concepts you may have forgotten Forward references will help you see the big picture.

BOX 9.5 The Hot Spice of Chili Peppers Biochemists now know the mechanism by which spice from

“hot” peppers exerts its action, causing a burning pain The active factor in capsicum peppers is a lipophilic vanilloid compound called capsaicin.

pain Control of the capsaicin receptor might have value in the relief of chronic pain in conditions such as arthritis.

H 3 CO HO

N

Capsaicin O

A nerve-cell protein receptor that responds to capsaicin has been identified and characterized It is an ion channel, and its amino acid sequence suggests that it has six trans- membrane domains Activation of the receptor by capsaicin causes the channel to open so that calcium and sodium ions can flow into the nerve cell and send an impulse to the brain.

The receptor is activated not only by vanilloid spices but also

by rapid increases in temperature In fact, the probable in vivo

role of the receptor is detection of heat Whereas the action of opioids suppresses pain, the action of vanilloids produces

bioengineering There are

many interesting stories to

tell, you’ll find them boxed

off from the main text so

they don’t distract you from

the main topic They will

give you insight to the

commercial, practical and

medical applications of

biochemistry.

360 CHAPTER 12 ■ Gluconeogenesis, The Pentose Phosphate Pathway, and Glycogen Metabolism

pyruvate carboxylase catalyzes the conversion of pyruvate to oxaloacetate The action is coupled to the hydrolysis of one molecule of ATP.

re-Pyruvate carboxylase is a large, complex enzyme composed of four identical units Each subunit has a biotin prosthetic group covalently linked to a lysine resi- due The biotin is required for the addition of bicarbonate to pyruvate Pyruvate carboxylase catalyzes a metabolically irreversible reaction and can be allosterically activated by acetyl CoA This is the only regulatory mechanism known for the en- zyme Accumulation of acetyl CoA indicates that it is not being efficiently metab- olized by the citric acid cycle Pyruvate carboxylase is stimulated in order to direct pyruvate to oxaloacetate instead of acetyl CoA Oxaloacetate can enter the citric acid cycle or serve as a precursor for glucose biosynthesis.

sub-Bicarbonate is one of the substrates in Reaction 12.2 sub-Bicarbonate is formed when carbon dioxide dissolves in water and the reaction is sometimes written with

as a substrate The pyruvate carboxylase reaction plays an important role in fixing carbon dioxide in bacteria and some eukaryotes This role may not be so ob- vious when we examine gluconeogenesis since the carbon dioxide is released in the very next reaction However, much of the oxaloacetate that is made is not used for gluconeogenesis Instead it replenishes the pool of citric acid cycle intermediates that serve as precursors to the biosynthesis of amino acids and lipids (Section 13.7).

is most often affected by controls at the level of transcription of its gene The level

of PEPCK activity in cells influences the rate of gluconeogenesis This is cially true in mammals in which gluconeogenesis occurs primarily in cells of the liver, kidneys, and small intestine During fasting in mammals, prolonged release

espe-of glucagon from the pancreas leads to an elevation espe-of intracellular cAMP, which triggers increased transcription of the PEPCK gene in the liver and increased syn- monal induction After several hours, the amount of PEPCK rises, and the rate of gluconeogenesis increases Insulin, abundant in the fed state, acts in opposition to glucagon at the level of the gene, reducing the rate of synthesis of PEPCK.

The reaction mechanism for pyruvate boxylase was described in Section 7.9.

car-Pyruvate

+

2

C O COO

COO Bicarbonate

Oxaloacetate 3

C O COO

COO Oxaloacetate

Phosphoenolpyruvate (PEP)

Phosphoenolpyruvate carboxykinase (PEPCK)

2 C COO 3

(ATP) GTP (ADP) GDP

2

(12.3)

2 OPO

xxxiii

▲ Figures

Biochemistry is a three-dimensional science and we have placed great emphasis on helping you visualize abstract concepts and molecules too small to see We have taken advantage of new rendering technologies to make illustrative figures both informative and beautiful For additional views

of molecules in 3-D go to the web site at www.prenhall.com/horton and

view the many PDB files.

End-of-Chapter Problems

Beginning with chapter 2, a number of

biochemical puzzles are included that

can be solved using the information

contained in the chapter Answers are

found at the back of the book.

If you want some additional practice,

we recommend you get a copy of The

Biochemistry Student Companion

by Allen Scism This book features

hundreds of additional problems,

including drill questions and chapter

review exercises.

Sialic acid CHOH

CH 2 OH

OH OH H

H

COOH O 7 2 3

6 5 4

1 8 9

Problems

1 Identify each of the following:

(a) Two aldoses whose configuration at carbons 3, 4, and

5 matches that of D -fructose.

(b) The enantiomer of D -galactose.

(c) An epimer of D -galactose that is also an epimer of

D -mannose.

(d) A ketose that has no chiral centers.

(e) A ketose that has only one chiral center.

(f) Monosaccharide residues of cellulose, amylose, and glycogen.

(g) Monosaccharide residues of chitin.

2 Draw Fischer projections for (a) L -mannose, (b) L -fucose (6-deoxy- L -galactose), (c) D -xylitol, and (d) D -iduronate.

3 Describe the general structural features of

glycos-aminoglycans.

4 Honey is an emulsion of microcrystalline D -fructose and

D -glucose Although D -fructose in polysaccharides exists mainly in the furanose form, solution or crystalline D - fructose (as in honey) is a mixture of several forms with

- D -fructopyranose (67%) and - D -fructofuranose (25%) predominating Draw the Fischer projection for D -fructose and show how it can cyclize to form both of the cyclized forms above.

5 Sialic acid D -neuraminic acid) is often found

in N-linked oligosaccharides that are involved in cell-cell

interactions Cancer cells synthesize much greater amounts

of sialic acid than normal cells and derivatives of sialic acid have been proposed as anticancer agents to block cell- surface interactions between normal and cancerous cells.

(N-Acetyl-a-b b

6 How many stereoisomers are possible for glucopyranose

and for fructofuranose? How many are D sugars in each case, and how many are L sugars?

7 Draw the structure of each of the following molecules and

label each chiral carbon with an asterisk.

(a) - D -Glucose 1-phosphate.

(b) 2-Deoxy- - D -ribose 5-phosphate.

(c) D -Glyceraldehyde 3-phosphate.

(d) L -Glucuronate.

8 In aqueous solution, virtually all D -glucose molecules are in the pyranose form Other aldoses have a 1799%2

b a

Answer the following questions about the structure of sialic acid.

(a) Is it an or a anomeric form?

(b) Will sialic acid mutorotate between and

anomer-ic forms?

(c) Is this a “deoxy” sugar?

(d) Will the open chain form of sialic acid be an hyde or a ketone?

alde-(e) How many chiral carbons are there in the sugar ring?

b a b

Special pair (P870) Bacteriochlorophyll a

Bacteriopheophytin

Quinone Q

QH2 2Hin

e

e e

e e

e e

α-carbon Carbonyl carbon Hydrogen Nitrogen Oxygen Side chain Right-handed α-helix Axis

0.15 nmRise (advance per amino acid residue)

0.54 nm Pitch (advance per turn)

Bacterial membrane

OUTSIDE (Periplasm)

INSIDE (Cytoplasm)

xxxiv

Companion Website

The new edition of the textbook offers a Companion Website at www.prenhall.com/horton This website is focused on the visualization of Biochemistry and includes hundreds of 3-D models of biomolecules, including nearly all of those that appear in your textbook Viewing these is a great way to ensure that you understand the key features of macromolecular architecture.

Also included on the Companion Website are MediaLabs that help you use the internet to practice and apply what you have learned With each MediaLab you are able to investigate the background of an important issue related to the chapter you have just read and then answer questions that test your comprehension of the topic MediaLab topics have been carefully chosen to focus on information that is timely and relevant in your daily life These investigations are designed to become a dynamic discovery activity in which you may participate and report, individually or in groups.

Instructor Resources

Test Item File (0-13-147601-7), by William Coleman, University of

Hartford and Donna Gosnell, Valdosta State University.

Provides a selection of more than 1500 questions referenced to the text.

TestGen-EQ Computerized Testing Software (0-13-147602-5)

In addition to the printed test bank, the test questions are available as part of the TestGen-EQ testing software, a text specific testing program that can reside on a network for administering tests It allows instructors to view and edit questions, export the questions as tests, and print them out in a variety of formats.

Transparency Pack (0-13-147616-5)

Over 100 full-color acetates and masters available free to qualified adopters.

Instructor Resource Center on CD (0-13-133055-1)

Includes most of the art from the textbook in a format for easy presentation on-screen Also includes pre-built PowerPoint slides.

Course Management

Instructors can access Prentice Hall OneKey content by downloading

it to a school’s server in cartridge or epack format by visiting

www.prenhall.com/onekey, clicking “OneKey For Instructors”,

clicking “Getting Started”, and selecting the desired platform

Student Resources

Biochemistry Student Companion (0-13-147605-X), by Allen Scism.

Contains hundreds of additional, carefully constructed, short answer, multiple choice, and challenge problems for each chapter, comprehensive, step-by-step solutions to all problems, lists of abbreviations and tables of essential data.

Companion Website

An online student tool that includes 3-D modules to help visualize biochemistry and MediaLabs to investigate important issues related to a particular chapter Please visit the site at http://www.prenhall.com/horton.

xxxv

xxxviiPrinciples of

Biochemistry

(b)

CH2

(c)

2 (a) Glycerol is polar; it is not amphipathic; and it readily dissolves in water.

(b) Hexadecanoyl phosphate is polar; it is amphipathic, and it does not readily dissolve

in water but forms micelles

(c) Laurate is polar; it is amphipathic; and it does not readily dissolve in water butforms micelles

(d) Glycine is polar; it is not amphipathic; and it readily dissolves in water

3 There is a larger osmotic pressure inside the cells than outside because the molar

con-centration of solutes is much greater inside cells than outside This results in a diffusion

of water into cells, causing them to swell and burst

4 If the pH of a solution is below the of any given ionizable group, the predominant

species will be the one with the dissociable proton on that group If the pH of a solution

is above the of any given ionizable group, the predominant species will be the one

with the dissociable proton off of that group.

The ion-product constant of water relates the concentrations of and (Equation 2.6)

(b) Human blood plasma If the then

6.

O

CO

H

R′R

OH

7 The total buffer

The pH can be calculated from the and the concentrations given using theHenderson-Hasselbalch equation

8 The for the ionization of is 7.2 The Henderson-Hasselbalch equation(Equation 2.18) indicates that when the concentrations of the acidic form

and its conjugate base are equivalent, the pH is equal to the because thelog term is zero Therefore, mixing 50 milliliters of solution A with 50 mil-liliters of solution B gives a buffer of pH 7.2 Since the concentration of each solution is0.02 M, mixing equal volumes gives a buffer whose phosphate concentration is also0.02 M The reason why this is an effective buffer is that the final pH is at the value.This means that the buffer will resist changes in pH over a considerable range

9 (a) The effective range of a buffer is from approximately one pH unit below to one pH

unit above the The buffering range for MOPS is therefore 6.2–8.2, and thebuffering range for SHS is 4.5–6.5 Use the Henderson-Hasselbalch equation to cal-culate the ratios of basic to acidic species

(b) An SHS buffer solution at pH 6.5 contains a much greater proportion of jugate base relative to acid (10:1) than MOPS does (1:5) Therefore, an SHS buffer would more effectively maintain the pH upon addition of acid:

con-Conversely, a MOPS buffer at pH 6.5 contains agreater proportion of acid than SHS does; therefore, MOPS would more effectivelymaintain the pH upon addition of base:

[RCOO]

[RCOOH]

=101

pKa

Total buffer concentration = 0.25 M + 0.15 M = 0.4 M

species = [weak acid 1HA2] + [conjugate base 1A2]

HH

OHOH

Fully protonated

OO

HH

OHOH

Fully ionized(dianion)

O P

OO

HH

OHOH

Partially ionized(monoanion)

O P

OO