Harper's Illustrated Biochemistry 30th Edition PDF

Contents Preface xi S E C T I O N 10 Bioinformatics & Computational Biology 97 I Structures & Functions of Proteins & Enzymes 1...

Contents Preface xi S E C T I O N 10 Bioinformatics & Computational Biology 97 I Structures & Functions of Proteins & Enzymes 1 Peter J. Kennelly, PhD & Victor W. Rodwell, PhD 1 Biochemistry & Medicine 1 S E C T I O N III Victor W. Rodwell, PhD & Robert K. Murray, MD, PhD Bioenergetics 113 2 Water & pH 6 Peter J. Kennelly, PhD & Victor W. Rodwell, PhD 11 Bioenergetics: The Role of ATP 113 3 Amino Acids & Peptides 15 Kathleen M. Botham, PhD, DSc & Peter A. Mayes, PhD, DSc Peter J. Kennelly, PhD & Victor W. Rodwell, PhD 12 Biologic Oxidation 119 4 Proteins: Determination of Primary Kathleen M. Botham, PhD, DSc & Peter A. Mayes, PhD, DSc Structure 25 Peter J. Kennelly, PhD & Victor W. Rodwell, PhD 13 The Respiratory Chain & Oxidative Phosphorylation 126 5 Proteins: Higher Orders of Structure 36 Kathleen M. Botham, PhD, DSc & Peter A. Mayes, PhD, DSc Peter J. Kennelly, PhD & Victor W. Rodwell, PhD S E C T I O N IV S E C T I O N Metabolism of Enzymes: Kinetics, II Carbohydrates 139 Mechanism, Regulation, & Bioinformatics 51 14 Overview of Metabolism & the Provision 6 Proteins: Myoglobin & Hemoglobin 51 of Metabolic Fuels 139 Peter J. Kennelly, PhD & Victor W. Rodwell, PhD David A. Bender, PhD & Peter A. Mayes, PhD, DSc 7 Enzymes: Mechanism of Action 60 15 Carbohydrates of Physiological Peter J. Kennelly, PhD & Victor W. Rodwell, PhD Significance 152 David A. Bender, PhD & Peter A. Mayes, PhD, DSc 8 Enzymes: Kinetics 73 Peter J. Kennelly, PhD & Victor W. Rodwell, PhD 16 The Citric Acid Cycle: The Central Pathway of Carbohydrate, Lipid & Amino Acid 9 Enzymes: Regulation of Activities 87 Metabolism 161 Peter J. Kennelly, PhD & Victor W. Rodwell, PhD David A. Bender, PhD & Peter A. Mayes, PhD, DSc  CONTENTS 17 Glycolysis & the Oxidation of Pyruvate 168 28 Catabolism of Proteins & of Amino Acid David A. Bender, PhD & Peter A. Mayes, PhD, DSc Nitrogen 287 Victor W. Rodwell, PhD 18 Metabolism of Glycogen 176 David A. Bender, PhD & Peter A. Mayes, PhD, DSc 29 Catabolism of the Carbon Skeletons of Amino Acids 297 19 Gluconeogenesis & the Control of Blood Victor W. Rodwell, PhD Glucose 185 David A. Bender, PhD & Peter A. Mayes, PhD, DSc 30 Conversion of Amino Acids to Specialized Products 313 20 The Pentose Phosphate Pathway & Other Victor W. Rodwell, PhD Pathways of Hexose Metabolism 196 David A. Bender, PhD & Peter A. Mayes, PhD, DSc 31 Porphyrins & Bile Pigments 323 Victor W. Rodwell, PhD & Robert K. Murray, MD, PhD S E C T I O N V Metabolism of Lipids 211 S E C T I O N Structure, Function, & 21 Lipids of Physiologic Significance 211 Kathleen M. Botham, PhD, DSc & Peter A. Mayes, PhD, DSc VII Replication of Informational Macromolecules 339 32 Nucleotides 339 22 Oxidation of Fatty Acids: Ketogenesis 223 Victor W. Rodwell, PhD Kathleen M. Botham, PhD, DSc & Peter A. Mayes, PhD, DSc 33 Metabolism of Purine & Pyrimidine 23 Biosynthesis of Fatty Nucleotides 347 Acids & Eicosanoids 232 Victor W. Rodwell, PhD Kathleen M. Botham, PhD, DSc & Peter A. Mayes, PhD, DSc 34 Nucleic Acid Structure & Function 359 24 Metabolism of Acylglycerols P. Anthony Weil, PhD & Sphingolipids 245 Kathleen M. Botham, PhD, DSc & Peter A. Mayes, PhD, DSc 35 DNA Organization, Replication, & Repair 370 25 Lipid Transport & Storage 253 P. Anthony Weil, PhD Kathleen M. Botham, PhD, DSc & Peter A. Mayes, PhD, DSc 36 RNA Synthesis, Processing, 26 Cholesterol Synthesis, Transport, & Modification 394 & Excretion 266 P. Anthony Weil, PhD Kathleen M. Botham, PhD, DSc & Peter A. Mayes, PhD, DSc 37 Protein Synthesis & the Genetic Code 413 P. Anthony Weil, PhD S E C T I O N VI Metabolism of Proteins & Amino Acids 281 38 Regulation of Gene Expression 428 P. Anthony Weil, PhD 27 Biosynthesis of the Nutritionally 39 Molecular Genetics, Recombinant DNA, Nonessential Amino Acids 281 & Genomic Technology 451 Victor W. Rodwell, PhD P. Anthony Weil, PhD  CONTENTS S E C T I O N S E C T I O N Biochemistry of VIII Extracellular & Intracellular Communication 477 X Special Topics (B) 607 40 Membranes: Structure 49 Intracellular Traffic & Sorting of Proteins 607 & Function 477 Kathleen M. Botham , PhD, DSc & Robert K. Murray, MD, PhD Robert K. Murray, MD, PhD & P. Anthony Weil, PhD 50 The Extracellular Matrix 627 41 The Diversity of the Endocrine Kathleen M. Botham, PhD, DSc & Robert K. Murray, MD, PhD System 498 P. Anthony Weil, PhD 51 Muscle & the Cytoskeleton 647 Peter J. Kennelly, PhD & Robert K. Murray, MD, PhD 42 Hormone Action & Signal Transduction 518 52 Plasma Proteins & Immunoglobulins 668 P. Anthony Weil, PhD Peter J. Kennelly, PhD, Robert K. Murray, MD, PhD, Molly Jacob, MBBS, MD, PhD & Joe Varghese, MBBS, MD 53 Red Blood Cells 689 S E C T I O N Peter J. Kennelly, PhD & Robert K. Murray, MD, PhD IX Special Topics (A) 537 54 White Blood Cells 700 Peter J. Kennelly, PhD & Robert K. Murray, MD, PhD 43 Nutrition, Digestion, & Absorption 537 S E C T I O N David A. Bender, PhD & Peter A. Mayes , PhD, DSc 44 Micronutrients: Vitamins XI Special Topics (C) 711 & Minerals 546 David A. Bender, PhD 55 Hemostasis & Thrombosis 711 Peter L. Gross, MD, MSc, FRCP(C), Robert K. Murray, MD, PhD, P. Anthony Weil, PhD, & Margaret L. Rand, PhD 45 Free Radicals & Antioxidant Nutrients 564 David A. Bender, PhD 56 Cancer: An Overview 722 Molly Jacob, MBBS, MD, PhD, Joe Varghese, MBBS, MD, Robert K. Murray, MD, PhD & P. Anthony Weil, PhD 46 Glycoproteins 569 David A. Bender, PhD & Robert K. Murray, MD, PhD 57 Biochemical Case Histories 746 David A. Bender, PhD 47 Metabolism of Xenobiotics 583 David A. Bender, PhD & Robert K. Murray, MD, PhD 58 The Biochemistry of Aging 755 Peter J. Kennelly, PhD 48 Clinical Biochemistry 589 David A. Bender, PhD, Joe Varghese, MBBS, MD, Molly Jacob, MBBS, MD, PhD, & Robert K. The Answer Bank 771 Murray, MD, PhD Index 777  S E C T I O N Structures & Functions I of Proteins & Enzymes C H A P T E R Biochemistry & Medicine Victor W. Rodwell, PhD & Robert K. Murray, MD, PhD 1 O BJEC TIVES Understand the importance of the ability of cell-free extracts of yeast to ferment sugars, an observation that enabled discovery of the intermediates After studying this chapter, of fermentation, glycolysis, and other metabolic pathways. you should be able to: Appreciate the scope of biochemistry and its central role in the life sciences, and that biochemistry and medicine are intimately related disciplines. Appreciate that biochemistry integrates knowledge of the chemical processes in living cells with strategies to maintain health, understand disease, identify potential therapies, and enhance our understanding of the origins of life on earth. Describe how genetic approaches have been critical for elucidating many areas of biochemistry, and how the Human Genome Project has furthered advances in numerous aspects of biology and medicine. BIOMEDICAL IMPORTANCE BIOCHEMISTRY BEGAN WITH THE Biochemistry and medicine enjoy a mutually cooperative DISCOVERY THAT A CELLFREE relationship. Biochemical studies have illuminated many EXTRACT OF YEAST CAN FERMENT SUGAR aspects of health and disease, and the study of various aspects of health and disease has opened up new areas of biochem- istry. The medical relevance of biochemistry both in normal The knowledge that yeast can convert the sugars to ethyl and abnormal situations is emphasized throughout this book. alcohol predates recorded history. It was not, however, until Biochemistry makes significant contributions to the fields of the earliest years of the 20th century that this process led cell biology, physiology, immunology, microbiology, pharma- directly to the science of biochemistry. Despite his insightful cology, and toxicology, as well as the fields of inflammation, investigations of brewing and wine making, the great French cell injury, and cancer. These close relationships emphasize microbiologist Louis Pasteur maintained that the process of that life, as we know it, depends on biochemical reactions and fermentation could only occur in intact cells. His error was processes. shown in 1899 by the brothers Büchner, who discovered that 1 2 SECTION I Structures & Functions of Proteins & Enzymes fermentation can indeed occur in cell-free extracts. This reve- used to prepare, analyze, purify, and identify metabolites and the lation resulted from storage of a yeast extract in a crock of con- activities of natural and recombinant enzymes and their three- centrated sugar solution added as a preservative. Overnight, dimensional structures are discussed in the following chapters. the contents of the crock fermented, spilled over the labora- tory bench and floor, and dramatically demonstrated that fermentation can proceed in the absence of an intact cell. BIOCHEMISTRY & MEDICINE This discovery made possible a rapid and highly productive HAVE STIMULATED MUTUAL series of investigations in the early years of the 20th century that initiated the science of biochemistry. These investigations ADVANCES revealed the vital role of inorganic phosphate, ADP, ATP, and The two major concerns for workers in the health sciences— NAD(H), and ultimately identified the phosphorylated sugars and particularly physicians—are the understanding and and the chemical reactions and enzymes (Gk “in yeast”) that maintenance of health and the understanding and effective convert glucose to pyruvate (glycolysis) or to ethanol and CO2 treatment of disease. Biochemistry impacts both of these fun- (fermentation). Subsequent research in the 1930s and 1940s damental concerns, and the interrelationship of biochemistry identified the intermediates of the citric acid cycle and of urea and medicine is a wide, two-way street. Biochemical studies biosynthesis, and provided insight into the essential roles of have illuminated many aspects of health and disease, and certain vitamin-derived cofactors or “coenzymes” such as conversely, the study of various aspects of health and dis- thiamin pyrophosphate, riboflavin, and ultimately coenzyme A, ease has opened up new areas of biochemistry (Figure 1–1). coenzyme Q, and cobamide coenzymes. The 1950s revealed Knowledge of protein structure and function was necessary how complex carbohydrates are synthesized from, and broken to identify and understand the single difference in amino acid down to simple sugars, and delineated the pathways for biosyn- sequence between normal hemoglobin and sickle cell hemo- thesis of pentoses and the breakdown of amino acids and lipids. globin, and analysis of numerous variant sickle cell and other Animal models, perfused intact organs, tissue slices, cell hemoglobins has contributed significantly to our understand- homogenates and their subfractions, and purified enzymes ing of the structure and function both of normal hemoglobin all were used to isolate and identify metabolites and enzymes. and of other proteins. During the early 1900s the English phy- These advances were made possible by the development in sician Archibald Garrod studied patients with the relatively the late 1930s and early 1940s of techniques such as analytical rare disorders of alkaptonuria, albinism, cystinuria, and pen- ultracentrifugation, paper and other forms of chromatogra- tosuria and established that these conditions were genetically phy, and the post-World War II availability of radioisotopes, determined. Garrod designated these conditions as inborn principally 14C, 3H and 32P, as “tracers” to identify the interme- errors of metabolism. His insights provided a foundation for diates in complex pathways such as that leading to the biosyn- the development of the field of human biochemical genetics. thesis of cholesterol and other isoprenoids and the pathways of A more recent example was investigation of the genetic and amino acid biosynthesis and catabolism. X-ray crystallography molecular basis of familial hypercholesterolemia, a disease was then used to solve the three-dimensional structure, first of that results in early onset atherosclerosis. In addition to clari- myoglobin, and subsequently of numerous proteins, polynu- fying different genetic mutations responsible for this disease, cleotides, enzymes, and viruses including that of the common this provided a deeper understanding of cell receptors and cold. Genetic advances that followed the realization that DNA mechanisms of uptake, not only of cholesterol, but of how was a double helix include the polymerase chain reaction, and other molecules’ cross cell membranes. Studies of oncogenes transgenic animals or those with gene knockouts. The methods and tumor suppressor genes in cancer cells have directed Biochemistry Nucleic acids Proteins Lipids Carbohydrates Genetic Sickle cell Athero- Diabetes diseases anemia sclerosis mellitus Medicine FIGURE 11 A two-way street connects biochemistry and medicine. Knowledge of the biochemical topics listed above the green line of the diagram has clarified our understanding of the diseases shown below the green line. Conversely, analyses of the diseases have casted light on many areas of biochemistry. Note that sickle cell anemia is a genetic disease, and that both atherosclerosis and diabetes mellitus have genetic components.  CHAPTER 1 Biochemistry & Medicine 3 attention to the molecular mechanisms involved in the control Impact of the Human Genome Project of normal cell growth. These examples illustrate how the study of disease can open up areas of basic biochemical research. on Biochemistry, Biology, & Medicine Science provides physicians and other workers in health care Initially unanticipated rapid progress in the late 1990s in and biology with a foundation that impacts practice, stimulates sequencing the human genome led in mid-2000 to the curiosity, and promotes the adoption of scientific approaches announcement that over 90% of the genome had been for continued learning. So long as medical treatment is firmly sequenced. This effort was headed by the International Human grounded in the knowledge of biochemistry and other basic Genome Sequencing Consortium and by Celera Genomics, a sciences, the practice of medicine will have a rational basis private company. Except for a few gaps, the sequence of the capable of accommodating and adapting to new knowledge. entire human genome was completed in 2003, just 50 years after the description of the double-helical nature of DNA by Watson and Crick. The implications for biochemistry, medi- NORMAL BIOCHEMICAL cine, and indeed for all of biology, are virtually unlimited. PROCESSES ARE THE BASIS For example, the ability to isolate and sequence a gene and to investigate its structure and function by sequencing and “gene OF HEALTH knockout” experiments have revealed previously unknown genes and their products, and new insights have been gained Biochemical Research Impacts Nutrition concerning human evolution and procedures for identifying & Preventive Medicine disease-related genes. The World Health Organization (WHO) defines health as a Major advances in biochemistry and understanding state of “complete physical, mental, and social well-being and human health and disease continue to be made by mutation of not merely the absence of disease and infirmity.” From a bio- the genomes of model organisms such as yeast and of eukary- chemical viewpoint, health may be considered that situation otes such as the fruit fly Drosophila melanogaster and the round in which all of the many thousands of intra- and extracellular worm Caenorhabditis elegans. Each organism has a short gen- reactions that occur in the body are proceeding at rates com- eration time and can be genetically manipulated to provide mensurate with the organism’s survival under pressure from insight into the functions of individual genes. These advances both internal and external challenges. The maintenance of can potentially be translated into approaches that help humans health requires optimal dietary intake of a number of chemi- by providing clues to curing human diseases such as cancer cals, chief among which are vitamins, certain amino acids and Alzheimer disease. Figure 1–2 highlights areas that have and fatty acids, various minerals, and water. Understanding developed or accelerated as a direct result of progress made in nutrition depends to a great extent on knowledge of biochem- the Human Genome Project (HGP). New “-omics” fields have istry, and the sciences of biochemistry and nutrition share a blossomed, each of which focuses on comprehensive study of focus on these chemicals. Recent increasing emphasis on sys- the structures and functions of the molecules with which each tematic attempts to maintain health and forestall disease, or is concerned. Definitions of these -omics fields mentioned preventive medicine, includes nutritional approaches to the below appear in the Glossary of this chapter. The products of prevention of diseases such as atherosclerosis and cancer. genes (RNA molecules and proteins) are being studied using the techniques of transcriptomics and proteomics. A spec- tacular example of the speed of progress in transcriptomics Most Diseases Have a Biochemical Basis is the explosion of knowledge about small RNA molecules as Apart from infectious organisms and environmental pollut- regulators of gene activity. Other -omics fields include glycom- ants, many diseases are manifestations of abnormalities in ics, lipidomics, metabolomics, nutrigenomics, and pharma- genes, proteins, chemical reactions, or biochemical processes, cogenomics. To keep pace with the information generated, each of which can adversely affect one or more critical bio- bioinformatics has received much attention. Other related chemical functions. Examples of disturbances in human fields to which the impetus from the HGP has carried over are biochemistry responsible for diseases or other debilitating biotechnology, bioengineering, biophysics, and bioethics. conditions include electrolyte imbalance, defective nutrient Nanotechnology is an active area, which, for example, may ingestion or absorption, hormonal imbalances, toxic chemi- provide novel methods of diagnosis and treatment for cancer cals or biologic agents, and DNA-based genetic disorders. and other disorders. Stem cell biology is at the center of much To address these challenges, biochemical research continues current research. Gene therapy has yet to deliver the promise to be interwoven with studies in disciplines such as genetics, that it appears to offer, but it seems probable that ultimately cell biology, immunology, nutrition, pathology, and pharma- will occur. Many new molecular diagnostic tests have devel- cology. In addition, many biochemists are vitally interested oped in areas such as genetic, microbiologic, and immunologic in contributing to solutions to key issues such as the ultimate testing and diagnosis. Systems biology is also burgeoning. survival of mankind, and educating the public to support use The outcomes of research in the various areas mentioned above of the scientific method in solving environmental and other will impact tremendously the future of biology, medicine, and major problems that confront us. the health sciences. Synthetic biology offers the potential for 4 SECTION I Structures & Functions of Proteins & Enzymes Transcriptomics Proteomics Glycomics Lipidomics Metabolomics Nutrigenomics Pharmacogenomics Bioinformatics HGP (Genomics) Bioengineering Biotechnology Biophysics Bioethics Stem cell biology Gene therapy Nanotechnology Molecular diagnostics Systems biology Synthetic biology FIGURE 12 The Human Genome Project (HGP) has influenced many disciplines and areas of research. Biochemistry is not listed since it predates com- mencement of the HGP, but disciplines such as bioinformatics, genomics, glycomics, lipidomics, metabolomics, molecular diagnostics, proteomics, and transcriptomics are nevertheless active areas of biochemical research. creating living organisms, initially small bacteria, from genetic Genomic research on model organisms such as yeast, the fruit material in vitro that might carry out specific tasks such as fly D. melanogaster, and the round worm C. elegans provides cleansing petroleum spills. All of the above make the 21st cen- insight into understanding human diseases tury an exhilarating time to be directly involved in biology and medicine. REFERENCES SUMMARY Alberts B: Model organisms and human health. Science 2010;330:1724. Biochemistry is the science concerned with studying the Alberts B: Lessons from genomics. Science 2011;331:511. various molecules that occur in living cells and organisms, Cammack R, Attwood T, Campbell P, et al (editors): Oxford the individual chemical reactions and their enzyme catalysts, Dictionary of Biochemistry and Molecular Biology. 2nd ed. and the expression and regulation of each metabolic process. Oxford University Press, 2006. Because life depends on biochemical reactions, biochemistry Cooke M: Science for physicians. Science 2010;329:1573. has become the basic language of all biologic sciences. Feero WG, Guttmacher AE, Collins FS: Genomic medicine—an Despite the focus on human biochemistry in this text, updated primer. N Engl J Med 2010;362:2001. biochemistry concerns the entire spectrum of life forms, from Gibson DG, Glass JI, Lartigue C, et al: Creation of a bacterial relatively simple viruses and bacteria and plants to complex cell controlled by a chemically synthesized genome. Science eukaryotes such as human beings. 2010;329:52. Biochemistry, medicine and other health care disciplines Kornberg A: Centenary of the birth of modern biochemistry. FASEB are intimately related. Health in all species depends on a J 1997;11:1209. harmonious balance of the biochemical reactions occurring in Online Mendelian Inheritance in Man (OMIM): Center for Medical the body, while disease reflects abnormalities in biomolecules, Genetics, Johns Hopkins University & National Center for biochemical reactions, or biochemical processes. Biotechnology Information, National Library of Medicine. http://www.ncbi.nlm.nih.gov/omim/. Advances in biochemical knowledge have illuminated many Scriver CR, Beaudet AL, Valle D, et al (editors): The Metabolic and areas of medicine, and the study of diseases has often revealed Molecular Bases of Inherited Disease, 8th ed. McGraw-Hill, previously unsuspected aspects of biochemistry. 2001. Available online and updated as The Online Metabolic & Biochemical approaches are often fundamental in illuminating Molecular Bases of Inherited Disease at www.ommbid.com. the causes of diseases and in designing appropriate therapies, Weatherall DJ: Systems biology and red cells. N Engl J Med and various biochemical laboratory tests represent an integral 2011;364:376. component of diagnosis and monitoring of treatment. A sound knowledge of biochemistry and of other related basic disciplines is essential for the rational practice of medicine and GLOSSARY related health sciences. Bioengineering: The application of engineering to biology and Results of the HGP and of research in related areas will have medicine. a profound influence on the future of biology, medicine, and Bioethics: The area of ethics that is concerned with the application other health sciences. of moral and ethical principles to biology and medicine.  CHAPTER 1 Biochemistry & Medicine 5 Bioinformatics: The discipline concerned with the collection, Nanotechnology: The development and application to medicine and storage, and analysis of biologic data, mainly DNA and protein to other areas of devices such as nanoshells which are only a few sequences (see Chapter 10). nanometers in size (10−9 m = 1 nm). Biophysics: The application of physics and its techniques to biology Nutrigenomics: The systematic study of the effects of nutrients on and medicine. genetic expression and of the effects of genetic variations on the Biotechnology: The field in which biochemical, engineering, and metabolism of nutrients. other approaches are combined to develop biological products of Pharmacogenomics: The use of genomic information and use in medicine and industry. technologies to optimize the discovery and development of new Gene Therapy: Applies to the use of genetically engineered genes to drugs and drug targets. treat various diseases. Proteomics: The proteome is the complete complement of proteins Genomics: The genome is the complete set of genes of an organism, of an organism. Proteomics is the systematic study of the and genomics is the in-depth study of the structures and structures and functions of proteomes and their variations in functions of genomes. health and disease. Glycomics: The glycome is the total complement of simple and Stem Cell Biology: Stem cells are undifferentiated cells that have complex carbohydrates in an organism. Glycomics is the the potential to self-renew and to differentiate into any of systematic study of the structures and functions of glycomes such the adult cells of an organism. Stem cell biology concerns the as the human glycome. biology of stem cells and their potential for treating various Lipidomics: The lipidome is the complete complement of lipids diseases. found in an organism. Lipidomics is the in-depth study of the Synthetic Biology: The field that combines biomolecular techniques structures and functions of all members of the lipidome and of with engineering approaches to build new biological functions their interactions, in both health and disease. and systems. Metabolomics: The metabolome is the complete complement of Systems Biology: The field concerns complex biologic systems metabolites (small molecules involved in metabolism) present studied as integrated entities. in an organism. Metabolomics is the in-depth study of their Transcriptomics: The comprehensive study of the transcriptome, structures, functions, and changes in various metabolic states. the complete set of RNA transcripts produced by the genome Molecular Diagnostics: Refers to the use of molecular approaches during a fixed period of time. such as DNA probes to assist in the diagnosis of various biochemical, genetic, immunologic, microbiologic, and other medical conditions.  C H A P T E R Water & pH Peter J. Kennelly, PhD & Victor W. Rodwell, PhD 2 OBJEC TIVES Describe the properties of water that account for its surface tension, viscosity, liquid state at ambient temperature, and solvent power. After studying this chapter, Use structural formulas to represent several organic compounds that can serve you should be able to: as hydrogen bond donors or acceptors. Explain the role played by entropy in the orientation, in an aqueous environment, of the polar and nonpolar regions of macromolecules. Indicate the quantitative contributions of salt bridges, hydrophobic interactions, and van der Waals forces to the stability of macromolecules. Explain the relationship of pH to acidity, alkalinity, and the quantitative determinants that characterize weak and strong acids. Calculate the shift in pH that accompanies the addition of a given quantity of acid or base to the pH of a buffered solution. Describe what buffers do, how they do it, and the conditions under which a buffer is most effective under physiologic or other conditions. Illustrate how the Henderson-Hasselbalch equation can be used to calculate the net charge on a polyelectrolyte at a given pH. BIOMEDICAL IMPORTANCE the pH of extracellular fluid between 7.35 and 7.45. Suspected disturbances of acid-base balance are verified by measuring Water is the predominant chemical component of living the pH of arterial blood and the CO2 content of venous blood. organisms. Its unique physical properties, which include the Causes of acidosis (blood pH 7.45) may follow vomiting ecules, derive from water’s dipolar structure and exceptional of acidic gastric contents. capacity for forming hydrogen bonds. The manner in which water interacts with a solvated biomolecule influences the structure both of the biomolecule and of water itself. An excel- WATER IS AN IDEAL BIOLOGIC lent nucleophile, water is a reactant or product in many met- abolic reactions. Regulation of water balance depends upon SOLVENT hypothalamic mechanisms that control thirst, on antidiuretic hormone (ADH), on retention or excretion of water by the Water Molecules Form Dipoles kidneys, and on evaporative loss. Nephrogenic diabetes insipi- A water molecule is an irregular, slightly skewed tetrahedron dus, which involves the inability to concentrate urine or adjust with oxygen at its center (Figure 2–1). The two hydrogens and to subtle changes in extracellular fluid osmolarity, results from the unshared electrons of the remaining two sp3-hybridized the unresponsiveness of renal tubular osmoreceptors to ADH. orbitals occupy the corners of the tetrahedron. The 105° angle Water has a slight propensity to dissociate into hydroxide between the two hydrogen atoms differs slightly from the ideal ions and protons. The concentration of protons, or acidity, of tetrahedral angle, 109.5°. Ammonia is also tetrahedral, with a aqueous solutions is generally reported using the logarithmic 107° angle between its three hydrogens. The strongly electro- pH scale. Bicarbonate and other buffers normally maintain negative oxygen atom in a water molecule attracts electrons 6  CHAPTER 2 Water & pH 7 H CH3 CH2 O H O 2e H 2e H H CH3 CH2 O H O 105° CH2 CH3 H FIGURE 21 The water molecule has tetrahedral geometry. R R II C O H N away from the hydrogen nuclei, leaving them with a partial RI R III positive charge, while its two unshared electron pairs consti- FIGURE 23 Additional polar groups participate in hydrogen tute a region of local negative charge. bonding. Shown are hydrogen bonds formed between alcohol and A molecule with electrical charge distributed asymmet- water, between two molecules of ethanol, and between the peptide rically about its structure is referred to as a dipole. Water’s carbonyl oxygen and the peptide nitrogen hydrogen of an adjacent amino acid. strong dipole is responsible for its high dielectric constant. As described quantitatively by Coulomb’s law, the strength of interaction F between oppositely charged particles is inversely Hydrogen bonding enables water to dissolve many organic proportionate to the dielectric constant ε of the surrounding biomolecules that contain functional groups which can par- medium. The dielectric constant for a vacuum is essentially ticipate in hydrogen bonding. The oxygen atoms of aldehydes, unity; for hexane it is 1.9; for ethanol, 24.3; and for water ketones, and amides, for example, provide lone pairs of elec- at 25°C, 78.5. Water therefore greatly decreases the force of trons that can serve as hydrogen acceptors. Alcohols, carbox- attraction between charged and polar species relative to water- ylic acids, and amines can serve both as hydrogen acceptors free environments with lower dielectric constants. Its strong and as donors of unshielded hydrogen atoms for formation of dipole and high dielectric constant enable water to dissolve hydrogen bonds (Figure 2–3). large quantities of charged compounds such as salts. Water Molecules Form Hydrogen Bonds INTERACTION WITH WATER A partially unshielded hydrogen nucleus covalently bound to an electron-withdrawing oxygen or nitrogen atom can INFLUENCES THE STRUCTURE interact with an unshared electron pair on another oxygen or OF BIOMOLECULES nitrogen atom to form a hydrogen bond. Since water mole- cules contain both of these features, hydrogen bonding favors Covalent and Noncovalent Bonds the self-association of water molecules into ordered arrays (Figure 2–2). Hydrogen bonding profoundly influences the Stabilize Biologic Molecules physical properties of water and accounts for its relatively high The covalent bond is the strongest force that holds molecules viscosity, surface tension, and boiling point. On average, each together (Table 2–1). Noncovalent forces, while of lesser mag- molecule in liquid water associates through hydrogen bonds nitude, make significant contributions to the structure, stabil- with 3.5 others. These bonds are both relatively weak and ity, and functional competence of macromolecules in living transient, with a half-life of a few picoseconds. Rupture of a hydrogen bond in liquid water requires only about 4.5 kcal/ mol, less than 5% of the energy required to rupture a covalent TABLE 21 Bond Energies for Atoms of Biologic O—H bond. Significance Energy Energy H H H H Bond Type (kcal/mol) Bond Type (kcal/mol) O O H O[O 34 O:O 96 H H H O H S[S 51 C[H 99 O O H O H H C[N 70 C:S 108 H O H S[H 81 O[H 110 FIGURE 22 Water molecules self-associate via hydrogen C[C 82 C:C 147 bonds. Shown are the association of two water molecules (left) and C[O 84 C:N 147 a hydrogen-bonded cluster of four water molecules (right). Notice that water can serve simultaneously both as a hydrogen donor and N[H 94 C:O 164 as a hydrogen acceptor. 8 SECTION I Structures & Functions of Proteins & Enzymes cells. These forces, which can be either attractive or repulsive,.50 Interaction energy (kcaI mol–1) involve interactions both within the biomolecule and between it and the water that forms the principal component of the sur-.25 rounding environment. 0 Biomolecules Fold to Position Polar & A –0.25 Charged Groups on Their Surfaces Most biomolecules are amphipathic; that is, they possess –0.50 regions rich in charged or polar functional groups as well 3.0 4.0 5.0 6.0 7.0 8.0 as regions with hydrophobic character. Proteins tend to fold R (Å) with the R-groups of amino acids with hydrophobic side chains in the interior. Amino acids with charged or polar FIGURE 24 The strength of van der Waals interactions var- amino acid side chains (eg, arginine, glutamate, serine, see ies with the distance, R, between interacting species. The force of interaction between interacting species increases with decreasing Table 3–1) generally are present on the surface in contact with distance between them until they are separated by the van der Waals water. A similar pattern prevails in a phospholipid bilayer contact distance (see arrow marked A). Repulsion due to interaction where the charged “head groups” of phosphatidylserine or between the electron clouds of each atom or molecule then super- phosphatidylethanolamine contact water while their hydro- venes. While individual van der Waals interactions are extremely phobic fatty acyl side chains cluster together, excluding water weak, their cumulative effect is nevertheless substantial for macro- molecules such as DNA and proteins which have many atoms in (see Figure 40–5). This pattern maximizes the opportunities close contact. for the formation of energetically favorable charge-dipole, dipole-dipole, and hydrogen bonding interactions between polar groups on the biomolecule and water. It also minimizes Electrostatic Interactions energetically unfavorable contacts between water and hydro- Interactions between charged groups help shape biomolecu- phobic groups. lar structure. Electrostatic interactions between oppositely charged groups within or between biomolecules are termed salt bridges. Salt bridges are comparable in strength to hydro- Hydrophobic Interactions gen bonds but act over larger distances. They therefore often Hydrophobic interaction refers to the tendency of nonpolar facilitate the binding of charged molecules and ions to pro- compounds to self-associate in an aqueous environment. This teins and nucleic acids. self-association is driven neither by mutual attraction nor by what are sometimes incorrectly referred to as “hydrophobic van der Waals Forces bonds.” Self-association minimizes the disruption of energeti- van der Waals forces arise from attractions between tran- cally favorable interactions between the surrounding water sient dipoles generated by the rapid movement of electrons molecules. of all neutral atoms. Significantly weaker than hydrogen While the hydrogens of nonpolar groups such as the bonds but potentially extremely numerous, van der Waals methylene groups of hydrocarbons do not form hydrogen forces decrease as the sixth power of the distance separat- bonds, they do affect the structure of the water that surrounds ing atoms (Figure 2–4). Thus, they act over very short dis- them. Water molecules adjacent to a hydrophobic group are tances, typically 2 to 4 Å. restricted in the number of orientations (degrees of freedom) that permit them to participate in the maximum number of energetically favorable hydrogen bonds. Maximal formation Multiple Forces Stabilize Biomolecules of multiple hydrogen bonds, which maximizes enthalpy, can The DNA double helix illustrates the contribution of mul- be maintained only by increasing the order of the adjacent tiple forces to the structure of biomolecules. While each water molecules, with an accompanying decrease in entropy. individual DNA strand is held together by covalent bonds, It follows from the second law of thermodynamics that the two strands of the helix are held together exclusively by the optimal free energy of a hydrocarbon-water mixture is a noncovalent interactions such as hydrogen bonds between function of both maximal enthalpy (from hydrogen bonding) nucleotide bases (Watson-Crick base pairing) and van der and highest entropy (maximum degrees of freedom). Thus, Waals interactions between the stacked purine and pyrimi- nonpolar molecules tend to form droplets that minimize dine bases. The double helix presents the charged phosphate exposed surface area and reduce the number of water mol- groups and polar hydroxyl groups from the ribose sugars ecules whose motional freedom becomes restricted. Similarly, of the DNA backbone to water while burying the relatively in the aqueous environment of the living cell the hydrophobic hydrophobic nucleotide bases inside. The extended backbone portions of biopolymers tend to be buried inside the structure maximizes the distance between negatively charged phos- of the molecule, or within a lipid bilayer, minimizing contact phates, minimizing unfavorable electrostatic interactions with water. (see Figure 34–2).  CHAPTER 2 Water & pH 9 WATER IS AN EXCELLENT the hydrolysis of ATP, a new coupled reaction can be gener- ated whose net overall change in free energy favors biopolymer NUCLEOPHILE synthesis. Metabolic reactions often involve the attack by lone pairs of elec- Given the nucleophilic character of water and its high trons residing on electron-rich molecules termed nucleophiles concentration in cells, why are biopolymers such as proteins upon electron-poor atoms called electrophiles. Nucleophiles and DNA relatively stable? And how can synthesis of biopoly- and electrophiles do not necessarily possess a formal negative mers occur in an aqueous environment that favors hydrolysis? or positive charge. Water, whose two lone pairs of sp3 electrons Central to both questions are the properties of enzymes. In bear a partial negative charge (see Figure 2–1), is an excellent the absence of enzymic catalysis, even reactions that are highly nucleophile. Other nucleophiles of biologic importance include favored thermodynamically do not necessarily take place rap- the oxygen atoms of phosphates, alcohols, and carboxylic acids; idly. Precise and differential control of enzyme activity and the the sulfur of thiols; and the nitrogen atom of amines and of the sequestration of enzymes in specific organelles determine the imidazole ring of histidine. Common electrophiles include the physiologic circumstances under which a given biopolymer carbonyl carbons in amides, esters, aldehydes, and ketones and will be synthesized or degraded. The ability of enzyme active the phosphorus atoms of phosphoesters. sites to sequester substrates in an environment from which Nucleophilic attack by water typically results in the cleav- water can be excluded facilitates biopolymer synthesis. age of the amide, glycoside, or ester bonds that hold biopoly- mers together. This process is termed hydrolysis. Conversely, Water Molecules Exhibit a Slight but when monomer units are joined together to form biopoly- Important Tendency to Dissociate mers, such as proteins or glycogen, water is a product, for The ability of water to ionize, while slight, is of central impor- example, during the formation of a peptide bond between two tance for life. Since water can act both as an acid and as a base, amino acids. its ionization may be represented as an intermolecular proton While hydrolysis is a thermodynamically favored reac- transfer that forms a hydronium ion (H3O+) and a hydroxide tion, the amide and phosphoester bonds of polypeptides and ion (OH−): oligonucleotides are stable in the aqueous environment of the cell. This seemingly paradoxical behavior reflects the fact H2O + H2O T H3O + OH− that the thermodynamics that govern the equilibrium point of a reaction do not determine the rate at which it will pro- The transferred proton is actually associated with a cluster of ceed toward its equilibrium point. In the cell, protein cata- water molecules. Protons exist in solution not only as H3O+, lysts called enzymes accelerate the rate of hydrolytic reactions but also as multimers such as H5O2+ and H7O3+. The proton is when needed. Proteases catalyze the hydrolysis of proteins nevertheless routinely represented as H+, even though it is in into their component amino acids, while nucleases catalyze fact highly hydrated. the hydrolysis of the phosphoester bonds in DNA and RNA. Since hydronium and hydroxide ions continuously recom- Careful control of the activities of these enzymes is required to bine to form water molecules, an individual hydrogen or oxygen ensure that they act only at appropriate times. cannot be stated to be present as an ion or as part of a water molecule. At one instant it is an ion; an instant later it is part Many Metabolic Reactions of a water molecule. Individual ions or molecules are therefore not considered. We refer instead to the probability that at any Involve Group Transfer instant in time a given hydrogen will be present as an ion or as Many of the enzymic reactions responsible for synthesis and part of a water molecule. Since 1 g of water contains 3.46 × 1022 breakdown of biomolecules involve the transfer of a chemical molecules, the ionization of water can be described statisti- group G from a donor D to an acceptor A to form an acceptor cally. To state that the probability that a hydrogen exists as an group complex, A—G: ion is 0.01 means that at any given moment in time, a hydro- D´G + A T A ´G + D gen atom has 1 chance in 100 of being an ion and 99 chances out of 100 of being part of a water molecule. The actual prob- The hydrolysis and phosphorolysis of glycogen, for example, ability of a hydrogen atom in pure water existing as a hydrogen involve the transfer of glucosyl groups to water or to ortho- ion is approximately 1.8 × 10−9. The probability of its being phosphate. The equilibrium constant for the hydrolysis of part of a water molecule thus is almost unity. Stated another covalent bonds strongly favors the formation of split products. way, for every hydrogen ion or hydroxide ion in pure water, Conversely, many group transfer reactions responsible for the there are 0.56 billion or 0.56 × 109 water molecules. Hydrogen biosynthesis of macromolecules involve the thermodynami- ions and hydroxide ions nevertheless contribute significantly cally unfavored formation of covalent bonds. Enzyme catalysts to the properties of water. play a critical role in surmounting these barriers by virtue of For dissociation of water, their capacity to directly link two normally separate reactions together. By linking an energetically unfavorable group transfer [H+ ][OH− ] K= reaction with a thermodynamically favorable reaction, such as [H2O] 10 SECTION I Structures & Functions of Proteins & Enzymes where the brackets represent molar concentrations (strictly 2. Calculate the base 10 logarithm of [H+]. speaking, molar activities) and K is the dissociation constant. 3. pH is the negative of the value found in step 2. Since 1 mole (mol) of water weighs 18 g, 1 liter (L) (1000 g) For example, for pure water at 25°C, of water contains 1000 ÷ 18 = 55.56 mol. Pure water thus is 55.56 molar. Since the probability that a hydrogen in pure pH = − log[H+ ] = − log10−7 = −(−7) = 7.0 water will exist as a hydrogen ion is 1.8 × 10−9, the molar con- centration of H+ ions (or of OH− ions) in pure water is the This value is also known as the power (English), puissant product of the probability, 1.8 × 10−9, times the molar concen- (French), or potennz (German) of the exponent, hence the use tration of water, 55.56 mol/L. The result is 1.0 × 10−7 mol/L. of the term “p.” We can now calculate the dissociation constant K for pure Low pH values correspond to high concentrations of H+ water: and high pH values correspond to low concentrations of H+. [H+ ][OH− ] [10−7 ][10−7 ] Acids are proton donors and bases are proton acceptors. K= = Strong acids (eg, HCl, H2SO4) completely dissociate into anions [H2O] [55.56] and protons even in strongly acidic solutions (low pH). Weak = 0.018 × 10−14 = 1.8 × 10−16 mol/L acids dissociate only partially in acidic solutions. Similarly, strong bases (eg, KOH, NaOH), but not weak bases like The molar concentration of water, 55.56 mol/L, is too great to Ca(OH)2, are completely dissociated even at high pH. Many be significantly affected by dissociation. It is therefore consid- biochemicals are weak acids. Exceptions include phosphory- ered to be essentially constant. This constant may therefore lated intermediates, whose phosphoryl group contains two be incorporated into the dissociation constant K to provide a dissociable protons, the first of which is strongly acidic. useful new constant Kw termed the ion product for water. The The following examples illustrate how to calculate the pH relationship between Kw and K is shown below: of acidic and basic solutions. [H+ ][OH− ] Example 1: What is the pH of a solution whose hydrogen K= = 1.8 × 10−16 mol/L [H2O] ion concentration is 3.2 × 10−4 mol/L? K w = ( K )[H2O] = [H+ ][OH− ] pH = − log[H+ ] = (1.8 × 10−16 mol/L)(55.56mol/L) = − log(3.2 × 10−4 ) = 1.00 × 10−14 (mol/L)2 = − log(3.2) − log(10−4 ) Note that the dimensions of K are moles per liter and those of = −0.5 + 4.0 Kw are moles2 per liter2. As its name suggests, the ion product = 3.5 Kw is numerically equal to the product of the molar concentra- tions of H+ and OH−: Example 2: What is the pH of a solution whose hydroxide + − ion concentration is 4.0 × 10−4 mol/L? We first define a quantity K w = [H ][OH ] pOH that is equal to −log[OH−] and that may be derived from the definition of Kw: At 25°C, Kw = (10−7)2, or 10−14 (mol/L)2. At temperatures below 25°C, Kw is somewhat less than 10−14, and at tempera- K w = [H+ ][OH− ] = 10−14 tures above 25°C it is somewhat greater than 10−14. Within the stated limitations of temperature, Kw equals 10−14 (mol/L)2 for Therefore, all aqueous solutions, even solutions of acids or bases. We use Kw to calculate the pH of acidic and basic solutions. log[H+ ] + log[OH− ] = log10−14 pH IS THE NEGATIVE LOG or pH + pOH = 14 OF THE HYDROGEN ION CONCENTRATION To solve the problem by this approach: The term pH was introduced in 1909 by Sörensen, who defined [OH− ] = 4.0 × 10−4 it as the negative log of the hydrogen ion concentration: pOH = − log[OH− ] + pH = − log[H ] = − log(4.0 × 10−4 ) This definition, while not rigorous, suffices for many bio- = − log(4.0) − log(10−4 ) chemical purposes. To calculate the pH of a solution: = −0.60 + 4.0 1. Calculate the hydrogen ion concentration [H+]. = 3.4  CHAPTER 2 Water & pH 11 Now We express the relative strengths of weak acids and bases in terms of their dissociation constants. Shown below are the pH = 14 − pOH = 14 − 3.4 expressions for the dissociation constant (Ka) for two repre- = 10.6 sentative weak acids, R—COOH and R—NH3+. The examples above illustrate how the logarithmic pH scale R ´ COOH T R ´ COO− + H+ facilitates recording and comparing hydrogen ion concentra- [R ´ COO− ][H+ ] tions that differ by orders of magnitude from one another, Ka = 0.00032 M (pH 3.5) and 0.000000000025 M (pH 10.6). [R ´ COOH] Example 3: What are the pH values of (a) 2.0 × 10−2 mol/L R ´ NH3+ T R ´ NH2 + H+ KOH and of (b) 2.0 × 10−6 mol/L KOH? The OH− arises from [R ´ NH2 ][H+ ] two sources, KOH and water. Since pH is determined by the Ka = total [H+] (and pOH by the total [OH−]), both sources must be [R ´ NH3+ ] considered. In the first case (a), the contribution of water to the total [OH−] is negligible. The same cannot be said for the Since the numeric values of Ka for weak acids are negative second case (b): exponential numbers, we express Ka as pKa, where pK a = − log K a Concentration (mol/L) Note that pKa is related to Ka as pH is to [H+]. The stronger the (a) (b) acid, the lower is its pKa value. Molarity of KOH 2.0 × 10−2 2.0 × 10−6 Representative weak acids (left), their conjugate bases (center), and pKa values (right) include the following: [OH−] from KOH 2.0 × 10−2 2.0 × 10−6 [OH−] from water 1.0 × 10−7 1.0 × 10−7 R ´ CH2 ´ COOH R ´ CH2COO− pK a = 4 − 5 − Total [OH ] 2.00001 × 10 −2 2.1 × 10 −6 R ´ CH2 ´ NH3+ R ´ CH2 ´ NH2 pK a = 9 − 10 − H2CO3 HCO3 pK a = 6.4 Once a decision has been reached about the significance of the − H2PO4 HPO−42 pK a = 7.2 contribution by water, pH may be calculated as above. The above examples assume that the strong base KOH is completely dissociated in solution and that the concentra- pKa is used to express the relative strengths of both acids and tion of OH− ions was thus equal to that due to the KOH plus bases. For any weak acid, its conjugate is a strong base. Similarly, that present initially in the water. This assumption is valid the conjugate of a strong base is a weak acid. The relative for dilute solutions of strong bases or acids, but not for weak strengths of bases are expressed in terms of the pKa of their bases or acids. Since weak electrolytes dissociate only slightly conjugate acids. For polyprotic compounds containing more in solution, we must use the dissociation constant to calcu- than one dissociable proton, a numerical subscript is assigned late the concentration of [H+] (or [OH−]) produced by a given to each dissociation, numbered starting from unity in decreas- molarity of a weak acid (or base) before calculating total [H+] ing order of relative acidity. For a dissociation of the type (or total [OH−]) and subsequently pH. R ´ NH+3 → R ´ NH2 + H+ Functional Groups That Are Weak Acids the pKa is the pH at which the concentration of the acid R— Have Great Physiologic Significance NH3+ equals that of the base R—NH2. From the above equations that relate Ka to [H+] and to the Many biochemicals possess functional groups that are weak concentrations of undissociated acid and its conjugate base, acids or bases. Carboxyl groups, amino groups, and phosphate when esters, whose second dissociation falls within the physiologic range, are present in proteins and nucleic acids, most coen- [R ´ COO− ] = [R ´ COOH] zymes, and most intermediary metabolites. Knowledge of the dissociation of weak acids and bases thus is basic to under- or when standing the influence of intracellular pH on structure and bio- [R ´ NH2 ] = [R ´ NH3+ ] logic activity. Charge-based separations such as electrophoresis and ion exchange chromatography are also best understood in then terms of the dissociation behavior of functional groups. We term the protonated species (HA or R—NH3+) the acid K a = [H+ ] and the unprotonated species (A− or R—NH2) its conjugate base. Similarly, we may refer to a base (A− or R—NH2) and its Thus, when the associated (protonated) and dissociated conjugate acid (HA or R—NH3+). (conjugate base) species are present at equal concentrations, 12 SECTION I Structures & Functions of Proteins & Enzymes the prevailing hydrogen ion concentration [H+] is numerically Inversion of the last term removes the minus sign and gives equal to the dissociation constant, Ka. If the logarithms of both the Henderson-Hasselbalch equation sides of the above equation are taken and both sides are multi- plied by −1, the expressions would be as follows: [A − ] pH = pK a + log [HA] K a = [H+ ] − log K a = − log[H+ ] The Henderson-Hasselbalch equation has great predictive value in protonic equilibria. For example, Since −log Ka is defined as pKa, and −log [H+] defines pH, the 1. When an acid is exactly half-neutralized, [A−] = [HA]. equation may be rewritten as Under these conditions, pK a = pH [A− ] ⎛1 ⎞ pH = pK a + log = pK a + log ⎜ ⎟ = pK a + 0 [HA] ⎝1 ⎠ that is, the pKa of an acid group is the pH at which the pro- tonated and unprotonated species are present at equal con- Therefore, at half-neutralization, pH = pKa. centrations. The pKa for an acid may be determined by adding 0.5 equivalent of alkali per equivalent of acid. The resulting 2. When the ratio [A−]/[HA] = 100:1, pH will equal the pKa of the acid. [A− ] pH = pK a + log [HA] The Henderson-Hasselbalch pH = pK a + log(100/1) = pK a + 2 Equation Describes the Behavior of Weak Acids & Buffers 3. When the ratio [A−]/[HA] = 1:10, The Henderson-Hasselbalch equation is derived below. pH = pK a + log(1/10) = pK a + (−1) A weak acid, HA, ionizes as follows: If the equation is evaluated at ratios of [A−]/[HA] ranging HA T H+ + A− from 103 to 10−3 and the calculated pH values are plotted, the The equilibrium constant for this dissociation is resulting graph describes the titration curve for a weak acid (Figure 2–5). [H+ ][A− ] Ka = [HA] Solutions of Weak Acids & Their Salts Buffer Changes in pH Cross-multiplication gives Solutions of weak acids or bases and their conjugates exhibit [H+ ][A− ] = K a [HA] buffering, the ability to resist a change in pH following addi- tion of strong acid or base. Many metabolic reactions are Divide both sides by [A−]: accompanied by the release or uptake of protons. Oxidative metabolism produces CO2, the anhydride of carbonic acid, [HA] [H+ ] = K a which if not buffered would produce severe acidosis. Biologic [A− ] maintenance of a constant pH involves buffering by phosphate, bicarbonate, and proteins, which accept or release protons to Take the log of both sides: ⎛ [HA] ⎞ log[H+ ] = log ⎜ K a − ⎟ ⎝ [A ] ⎠ 1.0 –1.0 meq of alkali added per meq of acid [HA] = log K a + log − 0.8 –0.8 [A ] Net charge 0.6 –0.6 Multiply through by −1: 0.4 –0.4 [HA] − log[H+ ] = − log K a − log − 0.2 –0.2 [A ] 0 0 Substitute pH and pKa for −log [H+] and −log Ka, respectively; 2 3 4 5 6 7 8 then pH [HA] FIGURE 25 Titration curve for an acid of the type HA. pH = pK a − log − [A ] The heavy dot in the center of the curve indicates the pKa, 5.0.  CHAPTER 2 Water & pH 13 resist a change in pH. For laboratory experiments using tissue TABLE 22 Relative Strengths of Selected Acids of extracts or enzymes, constant pH is maintained by the addi- Biologic Significance tion of buffers such as MES ([2-N-morpholino]-ethanesulfonic Monoprotic Acids acid, pKa 6.1), inorganic orthophosphate (pKa2 7.2), HEPES (N-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, pKa 6.8), Formic pK 3.75 or Tris (tris[hydroxymethyl]aminomethane, pKa 8.3). The value Lactic pK 3.86 of pKa relative to the desired pH is the major determinant of Acetic pK 4.76 which buffer is selected. Buffering can be observed by using a pH meter while Ammonium ion pK 9.25 titrating a weak acid or base (Figure 2–5). We can also cal- Diprotic Acids culate the pH shift that accompanies addition of acid or base Carbonic pK1 6.37 to a buffered solution. In the example below, the buffered solution (a weak acid, pKa = 5.0, and its conjugate base) is pK2 10.25 initially at one of four pH values. We will calculate the pH Succinic pK1 4.21 shift that results when 0.1 meq of KOH is added to 1 meq of pK2 5.64 each solution: Glutaric pK1 4.34 Initial pH 5.00 5.37 5.60 5.86 pK2 5.41 [A−]initial 0.50 0.70 0.80 0.88 Triprotic Acids [HA]initial 0.50 0.30 0.20 0.12 Phosphoric pK1 2.15 ([A−]/[HA])initial 1.00 2.33 4.00 7.33 pK2 6.82 Addition of 0.1 meq of KOH Produces pK3 12.38 Citric pK1 3.08 [A−]final 0.60 0.80 0.90 0.98 pK2 4.74 [HA]final 0.40 0.20 0.10 0.02 pK3 5.40 ([A−]/[HA])final 1.50 4.00 9.00 49.0 log ([A−]/[HA])final 0.18 0.60 0.95 1.69 Note: Tabulated values are the pKa values (-log of the dissociation constant) of selected monoprotic, diprotic, and triprotic acids. Final pH 5.18 5.60 5.95 6.69 ΔpH 0.18 0.60 0.95 1.69 succinic acid, which has two methylene groups between its carboxyl groups, is 5.6, whereas the second pKa for glutaric Notice that ΔpH, the change in pH per milliequivalent of acid, which has one additional methylene group, is 5.4. OH− added, depends on the initial pH. The solution resists changes in pH most effectively at pH values close to the pKa. A solution of a weak acid and its conjugate base buffers pKa Values Depend on the most effectively in the pH range pKa ± 1.0 pH unit. Properties of the Medium Figure 2–5 also illustrates how the net charge on one mole- The pKa of a functional group is also profoundly influenced cule of the acid varies with pH. A fractional charge of −0.5 does by the surrounding medium. The medium may either raise not mean that an individual molecule bears a fractional charge or lower the pKa relative to its value in water, depending on but that the probability is 0.5 that a given molecule has a unit whether the undissociated acid or its conjugate base is the negative charge at any given moment in time. Consideration charged species. The effect of dielectric constant on pKa may of the net charge on macromolecules as a function of pH pro- be observed by adding ethanol to water. The pKa of a carbox- vides the basis for separatory techniques such as ion exchange ylic acid increases, whereas that of an amine decreases because chromatography and electrophoresis (see Chapter 4). ethanol decreases the ability of water to solvate a charged spe- cies. The pKa values of dissociating groups in the interiors of Acid Strength Depends proteins thus are profoundly affected by their local environ- ment, including the presence or absence of water. on Molecular Structure Many acids of biologic interest possess more than one dissoci- ating group. The presence of local negative charge hinders pro- SUMMARY ton release from nearby acidic groups, raising their pKa. This Water forms hydrogen-bonded clusters with itself and with is illustrated by the pKa values of the three dissociating groups other proton donors or acceptors. Hydrogen bonds account for of phosphoric acid and citric acid (Table 2–2). The effect of the surface tension, viscosity, liquid state at room temperature, adjacent charge decreases with distance. The second pKa for and solvent power of water. 14 SECTION I Structures & Functions of Proteins & Enzymes Compounds that contain O or N can serve as hydrogen bond donors and/or acceptors. REFERENCES Reese KM: Whence came the symbol pH. Chem & Eng News Entropic forces dictate that macromolecules expose polar 2004;82:64. regions to an aqueous interface and bury nonpolar regions. Segel IM: Biochemical Calculations. Wiley, 1968. Salt bridges, hydrophobic interactions, and van der Waals Skinner JL: Following the motions of water molecules in aqueous forces participate in maintaining molecular structure. solutions. Science 2010;328:985. pH is the negative log of [H+]. A low pH characterizes an acidic Stillinger FH: Water revisited. Science 1980;209:451. solution, and a high pH denotes a basic solution. Suresh SJ, Naik VM: Hydrogen bond thermodynamic properties of The strength of weak acids is expressed by pKa, the negative water from dielectric constant data. J Chem Phys 2000;113:9727. log of the acid dissociation constant. Strong acids have low pKa Wiggins PM: Role of water in some biological processes. Microbiol values and weak acids have high pKa values. Rev 1990;54:432. Buffers resist a change in pH when protons are produced or consumed. Maximum buffering capacity occurs ±1 pH unit on either side of pKa. Physiologic buffers include bicarbonate, orthophosphate, and proteins.  C H A P T E R Amino Acids & Peptides Peter J. Kennelly, PhD & Victor W. Rodwell, PhD 3 OBJEC TIVES Diagram the structures and write the three- and one-letter designations for each of the amino acids present in proteins. After studying this chapter, Describe the contribution of each type of R group of the protein amino acids to you should be able to: their chemical properties. List additional key functions of amino acids and explain how certain amino acids in plant seeds can severely impact human health. Name the ionizable groups of the protein amino acids and list their approximate pKa values as free amino acids in aqueous solution. Calculate the pH of an unbuffered aqueous solution of a polyfunctional amino acid and the change in pH that occurs following the addition of a given quantity of strong acid or alkali. Define pI and explain its relationship to the net charge on a polyfunctional electrolyte. Explain how pH, pKa and pI can be used to predict the mobility of a polyelectrolyte, such as an amino acid, in a direct-current electrical field. Describe the directionality, nomenclature, and primary structure of peptides. Describe the conformational consequences of the partial double-bond character of the peptide bond and identify the bonds in the peptide backbone that are free to rotate. BIOMEDICAL IMPORTANCE are almost totally reabsorbed in the proximal tubule, conserv- In addition to providing the monomer units from which the ing them for protein synthesis and other vital functions. Not long polypeptide chains of proteins are synthesized, the l-α- all amino acids are, however, beneficial. While their proteins amino acids and their derivatives participate in cellular func- contain only l-α-amino acids, some microorganisms secrete tions as diverse as nerve transmission and the biosynthesis of mixtures of d-amino acids. Many bacteria elaborate peptides porphyrins, purines, pyrimidines, and urea. The neuroendo- that contain both d- and l-α-amino acids, several of which crine system employs short polymers of amino acids called possess therapeutic value, including the antibiotics bacitracin peptides as hormones, hormone-releasing factors, neuro- and gramicidin A and the antitumor agent bleomycin. Certain modulators, and neurotransmitters. Humans and other higher other microbial peptides are toxic. The cyanobacterial pep- animals cannot synthesize 10 of the l-α-amino acids present tides microcystin and nodularin are lethal in large doses, while in proteins in amounts adequate to support infant growth or small quantities promote the formation of hepatic tumors. to maintain adult health. Consequently, the human diet must The ingestion of certain amino acids present in the seeds of contain adequate quantities of these nutritionally essential legumes of the genus Lathyrus results in lathyrism, a tragic amino acids. Each day the kidneys filter over 50 g of free amino irreversible disease in which individuals lose control of their acids from the arterial renal blood. However, only traces of free limbs. Certain other plant seed amino acids have also been amino acids normally appear in the urine because amino acids implicated in neurodegenerative disease in natives of Guam. 15 16 SECTION I Structures & Functions of Proteins & Enzymes PROPERTIES OF AMINO ACIDS 5-hydroxylysine; the conversion of peptidyl glutamate to γ-carboxyglutamate; and the methylation, formylation, acety- The Genetic Code Specifies lation, prenylation, and phosphorylation of certain aminoacyl residues. These modifications significantly extend the biologic 20 L-`-Amino Acids diversity of proteins by altering their solubility, stability, cata- Although more than 300 amino acids occur in nature, pro- lytic activity, and interaction with other proteins. teins are synthesized almost exclusively from the set of 20 l-α- amino acids encoded by nucleotide triplets called codons (see Table 37–1). While the three-letter genetic code could Selenocysteine, the 21st potentially accommodate more than 20 amino acids, the Protein L-`-Amino Acid genetic code is redundant since several amino acids are Selenocysteine (Figure 3–1) is an l-α-amino acid found in specified by multiple codons. Scientists frequently represent proteins from every domain of life. Humans contain approxi- the sequences of peptides and proteins using one- and three- mately two dozen selenoproteins that include certain per- letter abbreviations for each amino acid (Table 3–1). These oxidases and reductases, selenoprotein P, which circulates in amino acids can be characterized as being either hydrophilic the plasma, and the iodothyronine deiodinases responsible or hydrophobic (Table 3–2), properties that affect their loca- for converting the prohormone thyroxine (T4) to the thyroid tion in a protein’s mature folded conformation (see Chapter 5). hormone 3,3'5-triiodothyronine (T3) (see Chapter 41). As its Some proteins contain additional amino acids that arise by name implies, a selenium atom replaces the sulfur of its ele- the post-translational modification of an amino acid already mental analog, cysteine. Selenocysteine is not the product of present in a peptide. Examples include the conversion of a posttranslational modification, but is inserted directly into peptidyl proline and peptidyl lysine to 4-hydroxyproline and a growing polypeptide during translation. Selenocysteine thus TABLE 31 L-`-Amino Acids Present in Proteins Name Symbol Structural Formula pK1 pK2 pK3 With Aliphatic Side Chains `-COOH `-NH3+ R Group Glycine Gly [G] H CH COO — 2.4 9.8 + NH3 Alanine Ala [A] 2.4 9.9 CH3 CH COO— NH3+ Valine Val [V] H 3C 2.2 9.7 —