Baixe Color Atlas of Biochemistry (Thieme, 2006) (476s) e outras Notas de estudo em PDF para Biomedicina, somente na Docsity! o AIRE of Biochemistry E E Es Eq E prad J. Koolman K. H. Roechm Second edition, reviced and enlargod Color Atlas of Biochemistry Second edition, revised and enlarged Jan Koolman Professor Philipps University Marburg Institute of Physiologic Chemistry Marburg, Germany Klaus-Heinrich Roehm Professor Philipps University Marburg Institute of Physiologic Chemistry Marburg, Germany 215 color plates by Juergen Wirth Thieme Stuttgart · New York VI Preface Biochemistry is a dynamic, rapidly growing field, and the goal of this color atlas is to illustrate this fact visually. The precise boun- daries between biochemistry and related fields, such as cell biology, anatomy, physiol- ogy, genetics, and pharmacology, are dif cult to define and, in many cases, arbitrary. This overlap is not coincidental. The object being studied is often the same—a nerve cell or a mitochondrion, for example—and only the point of view differs. For a considerable period of its history, bio- chemistry was strongly influenced by chem- istry and concentrated on investigating met- abolic conversions and energy transfers. Ex- plaining the composition, structure, and me- tabolism of biologically important molecules has always been in the foreground. However, new aspects inherited from biochemistry’s other parent, the biological sciences, are now increasingly being added: the relation- ship between chemical structure and biolog- ical function, the pathways of information transfer, observance of the ways in which biomolecules are spatially and temporally dis- tributed in cells and organisms, and an aware- ness of evolution as a biochemical process. These new aspects of biochemistry are bound to become more and more important. Owing to space limitations, we have concen- trated here on the biochemistry of humans and mammals, although the biochemistry of other animals, plants, and microorganisms is no less interesting. In selecting the material for this book, we have put the emphasis on subjects relevant to students of human med- icine. The main purpose of the atlas is to serve as an overview and to provide visual informa- tion quickly and ef ciently. Referring to text- books can easily fill any gaps. For readers encountering biochemistry for the first time, some of the plates may look rather complex. It must be emphasized, therefore, that the atlas is not intended as a substitute for a compre- hensive textbook of biochemistry. As the subject matter is often dif cult to vis- ualize, symbols, models, and other graphic elements had to be found that make compli- cated phenomena appear tangible. The graphics were designed conservatively, the aim being to avoid illustrations that might look too spectacular or exaggerated. Our goal was to achieve a visual and aesthetic way of representing scientific facts that would be simple and at the same time effective for teaching purposes. Use of graphics software helped to maintain consistency in the use of shapes, colors, dimensions, and labels, in par- ticular. Formulae and other repetitive ele- ments and structures could be handled easily and precisely with the assistance of the com- puter. Color-coding has been used throughout to aid the reader, and the key to this is given in two special color plates on the front and rear in- side covers. For example, in molecular models each of the more important atoms has a par- ticular color: gray for carbon, white for hydro- gen, blue for nitrogen, red for oxygen, and so on. The different classes of biomolecules are also distinguished by color: proteins are al- ways shown in brown tones, carbohydrates in violet, lipids in yellow, DNA in blue, and RNA in green. In addition, specific symbols are used for the important coenzymes, such as ATP and NAD+. The compartments in which biochemical processes take place are color- coded as well. For example, the cytoplasm is shown in yellow, while the extracellular space is shaded in blue. Arrows indicating a chem- ical reaction are always black and those rep- resenting a transport process are gray. In terms of the visual clarity of its presenta- tion, biochemistry has still to catch up with anatomy and physiology. In this book, we sometimes use simplified ball-and-stick mod- els instead of the classical chemical formulae. In addition, a number of compounds are rep- resented by space-filling models. In these cases, we have tried to be as realistic as pos- sible. The models of small molecules are based on conformations calculated by com- puter-based molecular modeling. In illustrat- ing macromolecules, we used structural infor- VIIPreface mation obtained by X-ray crystallography that is stored in the Protein Data Bank. In naming enzymes, we have followed the of - cial nomenclature recommended by the IUBMB. For quick identification, EC numbers (in italics) are included with enzyme names. To help students assess the relevance of the material (while preparing for an examination, for example), we have included symbols on the text pages next to the section headings to indicate how important each topic is. A filled circle stands for “basic knowledge,” a half- filled circle indicates “standard knowledge,” and an empty circle stands for “in-depth knowledge.” Of course, this classification only reflects our subjective views. This second edition was carefully revised and a significant number of new plates were added to cover new developments. We are grateful to many readers for their comments and valuable criticisms during the preparation of this book. Of course, we would also welcome further comments and sugges- tions from our readers. August 2004 Jan Koolman, Klaus-Heinrich Röhm Marburg Jürgen Wirth Darmstadt Contents Introduction . . . . . . . . . . . . . . . . . . . . 1 Basics Chemistry Periodic table. . . . . . . . . . . . . . . . . . . . 2 Bonds . . . . . . . . . . . . . . . . . . . . . . . . . 4 Molecular structure . . . . . . . . . . . . . . . 6 Isomerism . . . . . . . . . . . . . . . . . . . . . . 8 Biomolecules I . . . . . . . . . . . . . . . . . . . 10 Biomolecules II . . . . . . . . . . . . . . . . . . 12 Chemical reactions. . . . . . . . . . . . . . . . 14 Physical Chemistry Energetics . . . . . . . . . . . . . . . . . . . . . . 16 Equilibriums . . . . . . . . . . . . . . . . . . . . 18 Enthalpy and entropy. . . . . . . . . . . . . . 20 Reaction kinetics . . . . . . . . . . . . . . . . . 22 Catalysis . . . . . . . . . . . . . . . . . . . . . . . 24 Water as a solvent . . . . . . . . . . . . . . . . 26 Hydrophobic interactions . . . . . . . . . . . 28 Acids and bases . . . . . . . . . . . . . . . . . . 30 Redox processes. . . . . . . . . . . . . . . . . . 32 Biomolecules Carbohydrates Overview. . . . . . . . . . . . . . . . . . . . . . . 34 Chemistry of sugars . . . . . . . . . . . . . . . 36 Monosaccharides and disaccharides . . . 38 Polysaccharides: overview . . . . . . . . . . 40 Plant polysaccharides. . . . . . . . . . . . . . 42 Glycosaminoglycans and glycoproteins . 44 Lipids Overview. . . . . . . . . . . . . . . . . . . . . . . 46 Fatty acids and fats . . . . . . . . . . . . . . . 48 Phospholipids and glycolipids . . . . . . . 50 Isoprenoids . . . . . . . . . . . . . . . . . . . . . 52 Steroid structure . . . . . . . . . . . . . . . . . 54 Steroids: overview . . . . . . . . . . . . . . . . 56 Amino Acids Chemistry and properties. . . . . . . . . . . 58 Proteinogenic amino acids . . . . . . . . . . 60 Non-proteinogenic amino acids . . . . . . 62 Peptides and Proteins Overview. . . . . . . . . . . . . . . . . . . . . . . 64 Peptide bonds . . . . . . . . . . . . . . . . . . . 66 Secondary structures . . . . . . . . . . . . . . 68 Structural proteins . . . . . . . . . . . . . . . . 70 Globular proteins . . . . . . . . . . . . . . . . . 72 Protein folding . . . . . . . . . . . . . . . . . . . 74 Molecular models: insulin. . . . . . . . . . . 76 Isolation and analysis of proteins . . . . . 78 Nucleotides and Nucleic Acids Bases and nucleotides. . . . . . . . . . . . . . 80 RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Molecular models: DNA and RNA . . . . . 86 Metabolism Enzymes Basics. . . . . . . . . . . . . . . . . . . . . . . . . . 88 Enzyme catalysis . . . . . . . . . . . . . . . . . 90 Enzyme kinetics I . . . . . . . . . . . . . . . . . 92 Enzyme kinetics II . . . . . . . . . . . . . . . . 94 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . 96 Lactate dehydrogenase: structure . . . . . 98 Lactate dehydrogenase: mechanism . . . 100 Enzymatic analysis . . . . . . . . . . . . . . . . 102 Coenzymes 1 . . . . . . . . . . . . . . . . . . . . 104 Coenzymes 2 . . . . . . . . . . . . . . . . . . . . 106 Coenzymes 3 . . . . . . . . . . . . . . . . . . . . 108 Activated metabolites . . . . . . . . . . . . . . 110 Metabolic Regulation Intermediary metabolism . . . . . . . . . . . 112 Regulatory mechanisms . . . . . . . . . . . . 114 Allosteric regulation . . . . . . . . . . . . . . . 116 Transcription control . . . . . . . . . . . . . . 118 Hormonal control . . . . . . . . . . . . . . . . . 120 Energy Metabolism ATP . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Energetic coupling . . . . . . . . . . . . . . . . 124 Energy conservation at membranes. . . . 126 Photosynthesis: light reactions . . . . . . . 128 Photosynthesis: dark reactions . . . . . . . 130 Molecular models: membrane proteins . 132 Oxoacid dehydrogenases. . . . . . . . . . . . 134 Tricarboxylic acid cycle: reactions . . . . . 136 Tricarboxylic acid cycle: functions . . . . . 138 Respiratory chain . . . . . . . . . . . . . . . . . 140 ATP synthesis . . . . . . . . . . . . . . . . . . . . 142 Regulation . . . . . . . . . . . . . . . . . . . . . . 144 Respiration and fermentation . . . . . . . . 146 Fermentations . . . . . . . . . . . . . . . . . . . 148 VIII Introduction This paperback atlas is intended for students of medicine and the biological sciences. It provides an introduction to biochemistry, but with its modular structure it can also be used as a reference book for more detailed information. The 216 color plates provide knowledge in the field of biochemistry, ac- companied by detailed information in the text on the facing page. The degree of dif - culty of the subject-matter is indicated by symbols in the text: stands for “basic biochemical knowledge” indicates “standard biochemical knowl- edge” means “specialist biochemical knowledge.” Some general rules used in the structure of the illustrations are summed up in two ex- planatory plates inside the front and back covers. Keywords, definitions, explanations of unfamiliar concepts and chemical formulas can be found using the index. The book starts with a few basics in biochemistry (pp. 2–33). There is a brief explanation of the concepts and principles of chemistry (pp. 2–15). These include the periodic table of the elements, chemical bonds, the general rules governing molecular structure, and the structures of im- portant classes of compounds. Several basic concepts of physical chemistry are also essen- tial for an understanding of biochemical processes. Pages 16–33 therefore discuss the various forms of energy and their intercon- version, reaction kinetics and catalysis, the properties of water, acids and bases, and re- dox processes. These basic concepts are followed by a sec- tion on the structure of the important biomo- lecules (pp. 34–87). This part of the book is arranged according to the different classes of metabolites. It discusses carbohydrates, lipids, amino acids, peptides and proteins, nucleoti- des, and nucleic acids. The next part presents the reactions involved in the interconversion of these compounds—the part of biochemistry that is commonly referred to as metabolism (pp. 88–195). The section starts with a dis- cussion of the enzymes and coenzymes, and discusses the mechanisms of metabolic regu- lation and the so-called energy metabolism. After this, the central metabolic pathways are presented, once again arranged according to the class of metabolite (pp.150–195). The second half of the book begins with a discussion of the functional compartments within the cell, the cellular organelles (pp. 196–235). This is followed on pp. 236–265 by the current field of molecular genetics (molecular biology). A further extensive sec- tion is devoted to the biochemistry of individual tissues and organs (pp. 266–359). Here, it has only been possible to focus on the most important organs and organ systems— the digestive system, blood, liver, kidneys, muscles, connective and supportive tissues, and the brain. Other topics include the biochemistry of nutrition (pp. 360–369), the structure and function of important hormones (pp. 370–393), and growth and development (pp. 394–405). The paperback atlas concludes with a series of schematic metabolic “charts” (pp. 407–419). These plates, which are not accom- panied by explanatory text apart from a brief introduction on p. 406, show simplified ver- sions of the most important synthetic and degradative pathways. The charts are mainly intended for reference, but they can also be used to review previously learned material. The enzymes catalyzing the various reactions are only indicated by their EC numbers. Their names can be found in the systematically ar- ranged and annotated enzyme list (pp. 420–430). 1Chemistry Periodic table A. Biologically important elements There are 81 stable elements in nature. Fifteen of these are present in all living things, and a further 8–10 are only found in particular or- ganisms. The illustration shows the first half of the periodic table, containing all of the bio- logically important elements. In addition to physical and chemical data, it also provides information about the distribution of the ele- ments in the living world and their abun- dance in the human body. The laws of atomic structure underlying the periodic table are discussed in chemistry textbooks. More than 99% of the atoms in animals’ bodies are accounted for by just four ele- ments—hydrogen (H), oxygen (O), carbon (C) and nitrogen (N). Hydrogen and oxygen are the constituents of water, which alone makes up 60–70% of cell mass (see p.196). Together with carbon and nitrogen, hydrogen and oxy- gen are also the major constituents of the organic compounds on which most living processes depend. Many biomolecules also contain sulfur (S) or phosphorus (P). The above macroelements are essential for all or- ganisms. A second biologically important group of elements, which together represent only about 0.5% of the body mass, are present al- most exclusively in the form of inorganic ions. This group includes the alkali metals sodium (Na) and potassium (K), and the alkaline earth metals magnesium (Mg) and calcium (Ca). The halogen chlorine (Cl) is also always ionized in the cell. All other elements important for life are present in such small quantities that they are referred to as trace elements. These in- clude transition metals such as iron (Fe), zinc (Zn), copper (Cu), cobalt (Co) and manganese (Mn). A few nonmetals, such as iodine (I) and selenium (Se), can also be classed as essential trace elements. B. Electron configurations: examples The chemical properties of atoms and the types of bond they form with each other are determined by their electron shells. The elec- tron configurations of the elements are there- fore also shown in Fig. A. Fig. B explains the symbols and abbreviations used. More de- tailed discussions of the subject are available in chemistry textbooks. The possible states of electrons are called orbitals. These are indicated by what is known as the principal quantum number and by a letter—s, p, or d. The orbitals are filled one by one as the number of electrons increases. Each orbital can hold a maximum of two electrons, which must have oppositely directed “spins.” Fig. A shows the distribution of the electrons among the orbitals for each of the elements. For example, the six electrons of carbon (B1) occupy the 1s orbital, the 2s orbi- tal, and two 2p orbitals. A filled 1s orbital has the same electron configuration as the noble gas helium (He). This region of the electron shell of carbon is therefore abbreviated as “He” in Fig. A. Below this, the numbers of electrons in each of the other filled orbitals (2s and 2p in the case of carbon) are shown on the right margin. For example, the electron shell of chlorine (B2) consists of that of neon (Ne) and seven additional electrons in 3s and 3p orbitals. In iron (B3), a transition metal of the first series, electrons occupy the 4s orbital even though the 3d orbitals are still partly empty. Many reactions of the transition met- als involve empty d orbitals—e. g., redox reac- tions or the formation of complexes with bases. Particularly stable electron arrangements arise when the outermost shell is fully occu- pied with eight electrons (the “octet rule”). This applies, for example, to the noble gases, as well as to ions such as Cl– (3s23p6) and Na+ (2s22p6). It is only in the cases of hydrogen and helium that two electrons are already suf cient to fill the outermost 1s orbital. 2 Basics ?? ? ?? 1s 2s 2p 3s 3p 3d 4s 4p 4d 5s 5p 3d 4s 4d 5s 4 5 44.96 Sc 21 Ar 1 2 47.88 Ti 22 Ar 2 2 50.94 V 23 Ar 3 2 52.00 Cr 24 Ar 4 2 54.94 Mn 25 Ar 5 2 55.85 Fe 26 Ar 6 2 58.93 Co 27 Ar 7 2 58.69 Ni 28 Ar 8 2 63.55 Cu 29 Ar 9 2 65.39 Zn 30 Ar 10 2 3 4 5 6 7 8 9 10 11 12 1.01 H 1 1 63 4.00 He 2 2 6.94 Li 3 1 9.01 Be 2 4 10.81 B 5 2 1 12.01 C 6 He 2 2 14.01 N 7 He 2 3 1.4 16.00 O 8 He 2 4 25.5 19.00 F 9 He 2 5 20.18 He Ne 10 2 6 HeHe He 22.99 Ne Na 1 11 0.03 24.31 Mg 12 Ne 2 0.01 26.98 Ne Al 13 2 1 28.09 Si 14 Ne 2 2 30.97 Ne P 15 2 3 0.22 32.07 S 16 Ne 2 4 0.05 35.45 Cl 17 Ne 2 5 0.03 39.95 Ar 18 2 6 39.10 Ar K 19 1 0.06 40.08 Ar Ca 20 2 0.31 69.72 Ga 31 Ar 10 2 1 72.61 Ge 32 Ar 10 2 2 74.92 As 33 Ar 10 2 3 78.96 Se 34 Ar 10 79.90 Br Ar 10 2 5 83.80 Kr 36 Ar 10 2 6 126.9 I 53 Kr 10 2 5 2 4 35 Ne 1 2 3 4 5 1 2 13 14 15 16 17 18 30.97 P 15 0.22 ? Ne 2 3 9.5 95.94 Mo 42 Kr 4 2 s p ds p s p [Ne] [Ar] 4 3 2 1 3 2 1 4 3 2 1 3 2 1 [He] Alkaline earths Halogens Alkali metals Noble gases Group Relative atomic mass Chemical symbol Atomic number Electron configuration Percent (%) of human body all/most organisms Macro element Trace element Metal Semi-metal Non-metal Noble gas Group Pe rio d possibly for some Essential for... Boron group Nitrogen group Carbon group Oxygen group A. Biologically important elements B. Electron configurations: examples Helium (He, Noble gas) 1s2 Neon (Ne, Noble gas) 1s2 2s2 2p6 Argon (Ar, Noble gas) 1s2 2s2 2p6 3s2 3p6 1. Carbon (C) [He] 2s2 2p2 2. Chlorine (Cl) [Ne] 3s2 3p5 3. Iron (Fe) [Ar] 4s2 3d6 3Chemistry Molecular structure The physical and chemical behavior of mole- cules is largely determined by their constitu- tion (the type and number of the atoms they contain and their bonding). Structural formu- las can therefore be used to predict not only the chemical reactivity of a molecule, but also its size and shape, and to some extent its conformation (the spatial arrangement of the atoms). Some data providing the basis for such predictions are summarized here and on the facing page. In addition, L-dihy- droxyphenylalanine (L-dopa; see p. 352), is used as an example to show the way in which molecules are illustrated in this book. A. Molecule illustrations In traditional two-dimensional structural formulas (A1), atoms are represented as letter symbols and electron pairs are shown as lines. Lines between two atomic symbols symbolize two bonding electrons (see p. 4), and all of the other lines represent free electron pairs, such as those that occur in O and N atoms. Free electrons are usually not represented explic- itly (and this is the convention used in this book as well). Dashed or continuous circles or arcs are used to emphasize delocalized elec- trons. Ball-and-stick models (A2) are used to illus- trate the spatial structure of molecules. Atoms are represented as colored balls (for the color coding, see the inside front cover) and bonds (including multiple bonds) as gray cylinders. Although the relative bond lengths and angles correspond to actual conditions, the size at which the atoms are represented is too small to make the model more comprehensible. Space-filling van der Waals models (A3) are useful for illustrating the actual shape and size of molecules. These models represent atoms as truncated balls. Their effective ex- tent is determined by what is known as the van der Waals radius. This is calculated from the energetically most favorable distance be- tween atoms that are not chemically bonded to one another. B. Bond lengths and angles Atomic radii and distances are now usually expressed in picometers (pm; 1 pm = 10–12 m). The old angstrom unit (Å, Å = 100 pm) is now obsolete. The length of single bonds approximately corresponds to the sum of what are known as the covalent radii of the atoms involved (see inside front cover). Double bonds are around 10–20% shorter than single bonds. In sp3-hybridized atoms, the angle between the individual bonds is approx. 110°; in sp2-hybridized atoms it is approx. 120°. C. Bond polarity Depending on the position of the element in the periodic table (see p. 2), atoms have different electronegativity—i. e., a different tendency to take up extra electrons. The val- ues given in C2 are on a scale between 2 and 4. The higher the value, the more electronega- tive the atom. When two atoms with very different electronegativities are bound to one another, the bonding electrons are drawn toward the more electronegative atom, and the bond is polarized. The atoms involved then carry positive or negative partial charges. In C1, the van der Waals surface is colored according to the different charge con- ditions (red = negative, blue = positive). Oxy- gen is the most strongly electronegative of the biochemically important elements, with C=O double bonds being especially highly polar. D. Hydrogen bonds The hydrogen bond, a special type of nonco- valent bond, is extremely important in bio- chemistry. In this type of bond, hydrogen atoms of OH, NH, or SH groups (known as hydrogen bond donors) interact with free electrons of acceptor atoms (for example, O, N, or S). The bonding energies of hydrogen bonds (10–40 kJ mol–1) are much lower than those of covalent bonds (approx. 400 kJ mol–1). However, as hydrogen bonds can be very numerous in proteins and DNA, they play a key role in the stabilization of these molecules (see pp. 68, 84). The impor- tance of hydrogen bonds for the properties of water is discussed on p. 26. 6 Basics 0.9 2.1 2.5 3.0 3.5 4.0 1 2 3 4 H C N O FNa A H B A H B A H B 120° 120° 120° 120° 120° 110° 110° 110° 110° 110° 110° 108° 124 pm 11 1 pm 14 9 pm 110 pm 95 pm 154 pm 140 pm 137 pm 100 pm 270–280 pm 280 pm 290 pm 290 pm O C O C C N H H H H O O HH H H H HH O H H O O H H H H C CH N N O H H R1 H O N C HC C O R2 C C C N C N N HC N R H N H H H N C N CH CC O CH3 RO Chiral center 1. Formula illustration 2. Ball- and-stick model 3. Van der Waals model 1. Partial charges in L-dopa 2. Electronegativities C. Bond polarity B. Bond lengths and anglesA. Molecule illustrations D. Hydrogen bonds Increasing electronegativity Positive Neutral Negative Acid Base Initial state 1. Principle Donor Acceptor Hydrogen bond Dissociated acid Protonated base Complete reaction Water Proteins DNA 2. Examples 7Chemistry Isomerism Isomers are molecules with the same compo- sition (i. e. the same molecular formula), but with different chemical and physical proper- ties. If isomers differ in the way in which their atoms are bonded in the molecule, they are described as structural isomers (cf. citric acid and isocitric acid, D). Other forms of isomer- ism are based on different arrangements of the substituents of bonds (A, B) or on the presence of chiral centers in the molecule (C). A. cis–trans isomers Double bonds are not freely rotatable (see p. 4). If double-bonded atoms have different substituents, there are two possible orienta- tions for these groups. In fumaric acid, an intermediate of the tricarboxylic acid cycle (see p.136), the carboxy groups lie on different sides of the double bond (trans or E position). In its isomer maleic acid, which is not pro- duced in metabolic processes, the carboxy groups lie on the same side of the bond (cis or Z position). Cis–trans isomers (geometric isomers) have different chemical and physical properties—e. g., their melting points (Fp.) and pKa values. They can only be intercon- verted by chemical reactions. In lipid metabolism, cis–trans isomerism is particularly important. For example, double bonds in natural fatty acids (see p. 48) usually have a cis configuration. By contrast, unsatu- rated intermediates of β oxidation have a trans configuration. This makes the break- down of unsaturated fatty acids more compli- cated (see p.166). Light-induced cis–trans iso- merization of retinal is of central importance in the visual cycle (see p. 358). B. Conformation Molecular forms that arise as a result of rota- tion around freely rotatable bonds are known as conformers. Even small molecules can have different conformations in solution. In the two conformations of succinic acid illustrated opposite, the atoms are arranged in a similar way to fumaric acid and maleic acid. Both forms are possible, although conformation 1 is more favorable due to the greater distance between the COOH groups and therefore oc- curs more frequently. Biologically active mac- romolecules such as proteins or nucleic acids usually have well-defined (“native”) confor- mations, which are stabilized by interactions in the molecule (see p. 74). C. Optical isomers Another type of isomerism arises when a mol- ecule contains a chiral center or is chiral as a whole. Chirality (from the Greek cheir, hand) leads to the appearance of structures that behave like image and mirror-image and that cannot be superimposed (“mirror” iso- mers). The most frequent cause of chiral be- havior is the presence of an asymmetric C atom—i. e., an atom with four different sub- stituents. Then there are two forms (enan- tiomers) with different configurations. Usu- ally, the two enantiomers of a molecule are designated as L and D forms. Clear classifica- tion of the configuration is made possible by the R/S system (see chemistry textbooks). Enantiomers have very similar chemical properties, but they rotate polarized light in opposite directions (optical activity, see pp. 36, 58). The same applies to the enantiom- ers of lactic acid. The dextrorotatory L-lactic acid occurs in animal muscle and blood, while the D form produced by microorganisms is found in milk products, for example (see p.148). The Fischer projection is often used to represent the formulas for chiral centers (cf. p. 58). D. The aconitase reaction Enzymes usually function stereospecifically. In chiral substrates, they only accept one of the enantiomers, and the reaction products are usually also sterically uniform. Aconitate hydratase (aconitase) catalyzes the conver- sion of citric acid into the constitution isomer isocitric acid (see p.136). Although citric acid is not chiral, aconitase only forms one of the four possible isomeric forms of isocitric acid (2R,3S-isocitric acid). The intermediate of the reaction, the unsaturated tricarboxylic acid aconitate, only occurs in the cis form in the reaction. The trans form of aconitate is found as a constituent of certain plants. 8 Basics O N SP H O H O H CR H R' R O R' O C R R' O C H R' O PO O O R H O H CO H R' R O C O R' H O C O R' R O PO O O H H O PO O O R C R' O O PO O O R P O O O H N H R H N R'' R R' N H R R' R N C R' H O S H H S R H S R S R' O C S R' R N H H H A. Important classes of compounds Hemiacetal Carboxylic acid amide Phosphoric acid ester Thioester “energy-rich” bond Water Primary alcohol Ether Oxygen Secondary alcohol Amino group Nitrogen Primary amine Ammonia Tertiary amine Secondary amine Thiol Disulfide Sulfur Carboxylic acid ester Dihydrogen phosphate Ketone Aldehyde Carboxylic acid Phosphoric acid anhydride Mixed anhydride Carbonyl group Carboxyl group Hydrogen sulfide Sulfhydryl group Phosphorus Oxidation Oxidation Oxidation O H CH H R' 11Chemistry Biomolecules II Many biomolecules are made up of smaller units in a modular fashion, and they can be broken down into these units again. The con- struction of these molecules usually takes place through condensation reactions involv- ing the removal of water. Conversely, their breakdown functions in a hydrolytic fash- ion—i. e., as a result of water uptake. The page opposite illustrates this modular princi- ple using the example of an important coen- zyme. A. Acetyl CoA Coenzyme A (see also p.106) is a nucleotide with a complex structure (see p. 80). It serves to activate residues of carboxylic acids (acyl residues). Bonding of the carboxy group of the carboxylic acid with the thiol group of the coenzyme creates a thioester bond (-S-CO-R; see p.10) in which the acyl residue has a high chemical potential. It can therefore be trans- ferred to other molecules in exergonic reac- tions. This fact plays an important role in lipid metabolism in particular (see pp.162ff.), as well as in two reactions of the tricarboxylic acid cycle (see p.136). As discussed on p.16, the group transfer potential can be expressed quantitatively as the change in free enthalpy (∆G) during hy- drolysis of the compound concerned. This is an arbitrary determination, but it provides important indications of the chemical energy stored in such a group. In the case of acetyl- CoA, the reaction to be considered is: Acetyl CoA + H2O acetate + CoA In standard conditions and at pH 7, the change in the chemical potential G (∆G0, see p.18) in this reaction amounts to –32 kJ mol–1 and it is therefore as high as the ∆G0 of ATP hydrolysis (see p.18). In addition to the “energy-rich” thioester bond, acetyl-CoA also has seven other hydrolyzable bonds with dif- ferent degrees of stability. These bonds, and the fragments that arise when they are hydro- lyzed, will be discussed here in sequence. (1) The reactive thiol group of coenzyme A is located in the part of the molecule that is derived from cysteamine. Cysteamine is a bio- genic amine (see p. 62) formed by decarbox- ylation of the amino acid cysteine. (2) The amino group of cysteamine is bound to the carboxy group of another bio- genic amine via an acid amide bond (-CO- NH-). β-Alanine arises through decarboxyla- tion of the amino acid aspartate, but it can also be formed by breakdown of pyrimidine bases (see p.186). (3) Another acid amide bond (-CO-NH-) creates the compound for the next constituent, pantoinate. This compound con- tains a chiral center and can therefore appear in two enantiomeric forms (see p. 8). In natu- ral coenzyme A, only one of the two forms is found, the (R)-pantoinate. Human metabo- lism is not capable of producing pantoinate itself, and it therefore has to take up a compound of β-alanine and pantoinate— pantothenate (“pantothenic acid”)—in the form of a vitamin in food (see p. 366). (4) The hydroxy group at C-4 of pantoinate is bound to a phosphate residue by an ester bond. The section of the molecule discussed so far represents a functional unit. In the cell, it is produced from pantothenate. The molecule also occurs in a protein-bound form as 4- phosphopantetheine in the enzyme fatty acid synthase (see p.168). In coenzyme A, however, it is bound to 3,5-adenosine di- phosphate. (5) When two phosphate residues bond, they do not form an ester, but an “energy- rich” phosphoric acid anhydride bond, as also occurs in other nucleoside phosphates. By contrast, (6) and (7) are ester bonds again. (8) The base adenine is bound to C-1 of ribose by an N-glycosidic bond (see p. 36). In addition to C-2 to C-4, C-1 of ribose also rep- resents a chiral center. The -configuration is usually found in nucleotides. 12 Basics C H 3 C S O C H 2 C H 2 N C C H 2 H O C H 2 NH C C O H OH C C H 2 C H 3H 3C O P O OO P O OO C H 2 O H H H O OH H N N N HC N N H 2 P O OO Ribose A. Acetyl CoA Acetate Cysteamine β-Alanine Pantoinate Phosphate Phosphate Phosphate Thioester bond Acid–amide bond Phosphoric acid ester bond Phosphoric acid anhydride bond Van der Waals model Adenine Energy-rich bond Chiral centers Acid– amide bond Phosphoric acid ester bond Phosphoric acid ester bond N-glycosidic bond 13Chemistry Energetics To obtain a better understanding of the pro- cesses involved in energy storage and conver- sion in living cells, it may be useful first to recall the physical basis for these processes. A. Forms of work There is essentially no difference between work and energy. Both are measured in joule (J = 1 N m). An outdated unit is the calorie (1 cal = 4.187 J). Energy is defined as the abil- ity of a system to perform work. There are many different forms of energy—e. g., me- chanical, chemical, and radiation energy. A system is capable of performing work when matter is moving along a potential gra- dient. This abstract definition is best under- stood by an example involving mechanical work (A1). Due to the earth’s gravitational pull, the mechanical potential energy of an object is the greater the further the object is away from the center of the earth. A potential difference (∆P) therefore exists between a higher location and a lower one. In a waterfall, the water spontaneously follows this poten- tial gradient and, in doing so, is able to per- form work—e. g., turning a mill. Work and energy consist of two quantities: an intensity factor, which is a measure of the potential difference—i. e., the “driving force” of the process—(here it is the height differ- ence) and a capacity factor, which is a mea- sure of the quantity of the substance being transported (here it is the weight of the water). In the case of electrical work (A2), the intensity factor is the voltage—i. e., the electrical potential difference between the source of the electrical current and the “ground,” while the capacity factor is the amount of charge that is flowing. Chemical work and chemical energy are defined in an analogous way. The intensity factor here is the chemical potential of a mol- ecule or combination of molecules. This is stated as free enthalpy G (also known as “Gibbs free energy”). When molecules spon- taneously react with one another, the result is products at lower potential. The difference in the chemical potentials of the educts and products (the change in free enthalpy, G) is a measure of the “driving force” of the reac- tion. The capacity factor in chemical work is the amount of matter reacting (in mol). Although absolute values for free enthalpy G cannot be determined, ∆G can be calculated from the equilibrium constant of the reaction (see p.18). B. Energetics and the course of processes Everyday experience shows that water never flows uphill spontaneously. Whether a partic- ular process can occur spontaneously or not depends on whether the potential difference between the final and the initial state, ∆P = P2 – P1, is positive or negative. If P2 is smaller than P1, then ∆P will be negative, and the process will take place and perform work. Processes of this type are called exergonic (B1). If there is no potential difference, then the system is in equilibrium (B2). In the case of endergonic processes, ∆P is positive (B3). Processes of this type do not proceed sponta- neously. Forcing endergonic processes to take place requires the use of the principle of energetic coupling. This effect can be illustrated by a mechanical analogy (B4). When two masses M1 and M2 are connected by a rope, M1 will move upward even though this part of the process is endergonic. The sum of the two potential differences (∆Peff = ∆P1 + ∆P2) is the determining factor in coupled processes. When ∆Peff is negative, the entire process can proceed. Energetic coupling makes it possible to convert different forms of work and energy into one another. For example, in a flashlight, an exergonic chemical reaction provides an electrical voltage that can then be used for the endergonic generation of light energy. In the luminescent organs of various animals, it is a chemical reaction that produces the light. In the musculature (see p. 336), chemical en- ergy is converted into mechanical work and heat energy. A form of storage for chemical energy that is used in all forms of life is aden- osine triphosphate (ATP; see p.122). Ender- gonic processes are usually driven by cou- pling to the strongly exergonic breakdown of ATP (see p.122). 16 Basics J = Joule = N · m =1 kg · m2 · s-2, 1 cal = 4.187 J ∆P ∆P1 ∆P2 M1 M2 P3 P1 P2 P3 P1 P2 ∆ Peff ∆P · A. Forms of work Po te nt ia l Lo w er H ig he r Elevated position Lower position 1. Mechanical work 3. Chemical work Weight V ol ta ge Ground Charge Voltage source 2. Electrical work Quantity Products Educts C ha ng e in fr ee e ne rg y (∆ G ) H ei gh t ∆P < 0 Potential 1. Exergonic ∆P = 0 ∆P > 0 ∆Peff < 0 Potential 2. Equilibrium 3. Endergonic 4. Energetically coupled Coupled processes can occur spontaneously Form of work Mechanical Electrical Chemical Intensity factor Height Voltage Free-enthalpy change ∆G Unit m V = J · C -1 J · mol -1 Unit J · m -1 C mol Work = Height · Weight Voltage · Charge ∆G · Quantity Unit J J J Capacity factor Weight Charge Quantity B. Energetics and the course of processes Process occurs spontaneously Process cannot occur 17Physical Chemistry Equilibriums A. Group transfer reactions Every chemical reaction reaches after a time a state of equilibrium in which the forward and back reactions proceed at the same speed. The law of mass action describes the concentra- tions of the educts (A, B) and products (C, D) in equilibrium. The equilibrium constant K is di- rectly related to ∆G0, the change in free enthalpy G involved in the reaction (see p.16) under standard conditions (∆G0 = – R T ln K). For any given concentrations, the lower equation applies. At ∆G < 0, the reac- tion proceeds spontaneously for as long as it takes for equilibrium to be reached (i. e., until ∆G = 0). At ∆G > 0, a spontaneous reaction is no longer possible (endergonic case; see p.16). In biochemistry, ∆G is usually related to pH 7, and this is indicated by the “prime” symbol (∆G0 or ∆G). As examples, we can look at two group transfer reactions (on the right). In ATP (see p.122), the terminal phosphate residue is at a high chemical potential. Its transfer to water (reaction a, below) is therefore strongly exer- gonic. The equilibrium of the reaction (∆G = 0; see p.122) is only reached when more than 99.9% of the originally available ATP has been hydrolyzed. ATP and similar compounds have a high group transfer potential for phosphate residues. Quantita- tively, this is expressed as the G of hydrolysis (∆G0 = –32 kJ mol–1; see p.122). In contrast, the endergonic transfer of am- monia (NH3) to glutamate (Glu, reaction b, ∆G0 = +14 kJ mol–1) reaches equilibrium so quickly that only minimal amounts of the product glutamine (Gln) can be formed in this way. The synthesis of glutamine from these preliminary stages is only possible through energetic coupling (see pp.16, 124). B. Redox reactions The course of electron transfer reactions (re- dox reactions, see p.14) also follows the law of mass action. For a single redox system (see p. 32), the Nernst equation applies (top). The electron transfer potential of a redox system (i. e., its tendency to give off or take up elec- trons) is given by its redox potential E (in standard conditions, E0 or E0). The lower the redox potential of a system is, the higher the chemical potential of the transferred elec- trons. To describe reactions between two re- dox systems, ∆Ε—the difference between the two systems’ redox potentials—is usually used instead of ∆G. ∆G and ∆E have a simple relationship, but opposite signs (below). A redox reaction proceeds spontaneously when ∆E > 0, i. e. ∆G < 0. The right side of the illustration shows the way in which the redox potential E is depen- dent on the composition (the proportion of the reduced form as a %) in two biochemically important redox systems (pyruvate/lactate and NAD+/NADH+H+; see pp. 98, 104). In the standard state (both systems reduced to 50%), electron transfer from lactate to NAD+ is not possible, because ∆E is negative (∆E = –0.13 V, red arrow). By contrast, transfer can proceed successfully if the pyruvate/lactate system is reduced to 98% and NAD+/NADH is 98% oxi- dized (green arrow, ∆E = +0.08 V). C. Acid–base reactions Pairs of conjugated acids and bases are always involved in proton exchange reactions (see p. 30). The dissociation state of an acid–base pair depends on the H+ concentration. Usu- ally, it is not this concentration itself that is expressed, but its negative decadic logarithm, the pH value. The connection between the pH value and the dissociation state is described by the Henderson–Hasselbalch equation (be- low). As a measure of the proton transfer potential of an acid–base pair, its pKa value is used—the negative logarithm of the acid constant Ka (where “a” stands for acid). The stronger an acid is, the lower its pKa value. The acid of the pair with the lower pKa value (the stronger acid—in this case acetic acid, CH3COOH) can protonate (green arrow) the base of the pair with the higher pKa (in this case NH3), while ammonium acetate (NH4 + and CH3COO –) only forms very little CH3COOH and NH3. 18 Basics 1 2 3 4 5 6 1 2 3 4 5 6 ∆H = - 287 kJ · mol -1 ∆G = - 238 kJ · mol -1 -T · ∆S = +49 kJ · mol -1 -T · ∆S = - 12.8 kJ · mol -1 ∆G = - 9.0 kJ · mol -1 ∆H = +3.8 kJ · mol -1 O2 1 2 3 4 5 6 1 2 3 4 5 6 CO2 -200 -100 0 +100 +200 -12 -8 -4 0 +4 +8 +12 ∆G = ∆H - T · ∆S H2O A. Heat of reaction and calorimetry 1. “Knall-gas” reaction 2. Dissolution of NaCl in water Low degree of order System re- leases heat, ∆H <0 (exothermic) 1 mol H2O (liquid) Higher degree of order, ∆S < 0 Lower degree of order ∆S > 0 1 mol Na 1 mol Cl System absorbs heat, ∆H > 0 (endothermic) High degree of order Ignition wire to start the reaction Thermometer Temperature insulation Pressurized metal container Water Sample Stirrer Water heated An enthalpy of 1kJ warms 1 l of water by 0.24 ºC Combustion 1 mol H2 1 mol NaCl (crystalline) ∆H: change of enthalpy, heat exchange ∆S: change of entropy, i.e. degree of order Gibbs-Helmholtz equation 1/2 mol O2 B. Enthalpy and entropy Water Energy Energy 21Physical Chemistry Reaction kinetics The change in free enthalpy ∆G in a reaction indicates whether or not the reaction can take place spontaneously in given conditions and how much work it can perform (see p.18). However, it does not tell us anything about the rate of the reaction—i. e., its kinetics. A. Activation energy Most organic chemical reactions (with the exception of acid–base reactions) proceed only very slowly, regardless of the value of ∆G. The reason for the slow reaction rate is that the molecules that react—the educts—have to have a certain minimum en- ergy before they can enter the reaction. This is best understood with the help of an energy diagram (1) of the simplest possible reaction A B. The educt A and the product B are each at a specific chemical potential (Ge and Gp, respectively). The change in the free enthalpy of the reaction, ∆G, corresponds to the differ- ence between these two potentials. To be converted into B, A first has to overcome a potential energy barrier, the peak of which, Ga, lies well above Ge. The potential difference Ga –Ge is the activation energy Ea of the re- action (in kJ mol–1). The fact that A can be converted into B at all is because the potential Ge only represents the average potential of all the molecules. Individual molecules may occasionally reach much higher potentials—e. g., due to collisions with other molecules. When the increase in energy thus gained is greater than Ea, these molecules can overcome the barrier and be converted into B. The energy distribution for a group of molecules of this type, as calculated from a simple model, is shown in (2) and (3). ∆n/n is the fraction of molecules that have reached or exceeded energy E (in kJ per mol). At 27 °C, for example, approximately 10% of the molecules have energies > 6 kJ mol–1. The typical activation energies of chemical reactions are much higher. The course of the energy function at energies of around 50 kJ mol–1 is shown in (3). Statistically, at 27 °C only two out of 109 molecules reach this energy. At 37 °C, the figure is already four. This is the basis for the long-familiar “Q10 law”—a rule of thumb that states that the speed of biological processes approximately doubles with an increase in temperature of 10 °C. B. Reaction rate The velocity v of a chemical reaction is deter- mined experimentally by observing the change in the concentration of an educt or product over time. In the example shown (again a reaction of the A B type), 3 mmol of the educt A is converted per second and 3 mmol of the product B is formed per second in one liter of the solution. This corresponds to a rate of v = 3 mM s–1 = 3 10–3 mol L–1 s–1 C. Reaction order Reaction rates are influenced not only by the activation energy and the temperature, but also by the concentrations of the reactants. When there is only one educt, A (1), v is proportional to the concentration [A] of this substance, and a first-order reaction is in- volved. When two educts, A and B, react with one another (2), it is a second order reaction (shown on the right). In this case, the rate v is proportional to the product of the educt concentrations (12 mM2 at the top, 24 mM2 in the middle, and 36 mM2 at the bottom). The proportionality factors k and k are the rate constants of the reaction. They are not dependent on the reaction concentra- tions, but depend on the external conditions for the reaction, such as temperature. In B, only the kinetics of simple irreversible reactions is shown. More complicated cases, such as reaction with three or more reversible steps, can usually be broken down into first- order or second-order partial reactions and described using the corresponding equations (for an example, see the Michaelis–Menten reaction, p. 92). 22 Basics 5 0 s 0 s 1 s 10 15 1 2 3 1 2 3 C1 s 1. 2. 0 s 1 s 3 s 1. 2. 3. 0.0 0.5 1.0 0 5 10 0 2 4 6 8 10 55 50 45 1. 2. 3. [A] (mM) A. Activation energy B. Reaction rate mM = mmol · l-1 Product B Substrate A Chemical potential En er gy (k J · m ol -1 ) Activation energy En er gy (k J · m ol -1 ) First-order reaction Second-order reaction 1 Liter k = 1/5 s -1 k' = 1/12 l · mmol-1· s -1 k, k' : Rate constants v (mM · s-1) (mM) v (mM · s-1) v = k · [A] v = k' · [A] · [B] C. Reaction order Ea ∆G = Gp - Ge Ga - Ge= ∆n/n ∆n/n · 109 27 ˚C 27˚C 37˚C = 32 mM[A]0 = 3 mM[B]0 [A] = 23 mM ∆[A] = -9 mM [A] = 29 mM ∆[A] = -3 mM [B] = 6 mM ∆[B] = 3 mM [B] = 12 mM ∆[B] = 9 mM ∆t = 1 s ∆t = 3 s v = -∆ [A] / ∆t = ∆ [ B] / ∆t ( mol · l -1 · s -1 ) = 3[A] ˚ = 12[A] ˚= 1[B] ˚ = 6[A] ˚= 4[B] ˚ = 12[B] ˚ A C A B+ Ga Ge Gp 23Physical Chemistry Water as a solvent Life as we know it evolved in water and is still absolutely dependent on it. The properties of water are therefore of fundamental impor- tance to all living things. A. Water and methane The special properties of water (H2O) become apparent when it is compared with methane (CH4). The two molecules have a similar mass and size. Nevertheless, the boiling point of water is more than 250 °C above that of methane. At temperatures on the earth’s sur- face, water is liquid, whereas methane is gas- eous. The high boiling point of water results from its high vaporization enthalpy, which in turn is due to the fact that the density of the electrons within the molecule is unevenly distributed. Two corners of the tetrahedrally- shaped water molecule are occupied by un- shared electrons (green), and the other two by hydrogen atoms. As a result, the H–O–H bond has an angled shape. In addition, the O–H bonds are polarized due to the high elec- tronegativity of oxygen (see p. 6). One side of the molecule carries a partial charge (δ) of about –0.6 units, whereas the other is corre- spondingly positively charged. The spatial separation of the positive and negative charges gives the molecule the properties of an electrical dipole. Water molecules are therefore attracted to one another like tiny magnets, and are also connected by hydrogen bonds (B) (see p. 6). When liquid water vapor- izes, a large amount of energy has to be ex- pended to disrupt these interactions. By con- trast, methane molecules are not dipolar, and therefore interact with one another only weakly. This is why liquid methane vaporizes at very low temperatures. B. Structure of water and ice The dipolar nature of water molecules favors the formation of hydrogen bonds (see p. 6). Each molecule can act either as a donor or an acceptor of H bonds, and many molecules in liquid water are therefore connected by H bonds (1). The bonds are in a state of constant fluctuation. Tetrahedral networks of mole- cules, known as water “clusters,” often arise. As the temperature decreases, the proportion of water clusters increases until the water begins to crystallize. Under normal atmo- spheric pressure, this occurs at 0 °C. In ice, most of the water molecules are fixed in a hexagonal lattice (3). Since the distance be- tween the individual molecules in the frozen state is on average greater than in the liquid state, the density of ice is lower than that of liquid water. This fact is of immense biological importance—it means, for example, that in winter, ice forms on the surface of open stretches of water first, and the water rarely freezes to the bottom. C. Hydration In contrast to most other liquids, water is an excellent solvent for ions. In the electrical field of cations and anions, the dipolar water molecules arrange themselves in a regular fashion corresponding to the charge of the ion. They form hydration shells and shield the central ion from oppositely charged ions. Metal ions are therefore often present as hexahydrates ([Me(H2O)6 2+], on the right). In the inner hydration sphere of this type of ion, the water molecules are practically immobi- lized and follow the central ion. Water has a high dielectric constant of 78—i. e., the elec- trostatic attraction force between ions is re- duced to 1/78 by the solvent. Electrically charged groups in organic molecules (e. g., carboxylate, phosphate, and ammonium groups) are also well hydrated and contribute to water solubility. Neutral molecules with several hydroxy groups, such as glycerol (on the left) or sugars, are also easily soluble, because they can form H bonds with water molecules. The higher the proportion of polar functional groups there is in a molecule, the more water-soluble (hydrophilic) it is. By con- trast, molecules that consist exclusively or mainly of hydrocarbons are poorly soluble or insoluble in water. These compounds are called hydrophobic (see p. 28). 26 Basics HO HO HO 1. 2. 3. H H H H H H Density 0.92 g · cm-3 hexagonal lattice, stabilized by hydrogen bonds Ice Ethanol A. Water and methane B. Structure of water and ice Anion Cation Glycerol [Me (H2O)6] 2 δ +0.3 δ -0.6 δ +0.3 H2O CH4 18 Da 16 Da +100 °C -162 °C 41 8 6.2 0 Molecular mass Boiling point Heat of vaporization (kJ · mol-1) Dipole moment (10-30 C · m) Water (H2O) Methane (CH4) C. Hydration density 1.00 g · cm-3 short-lived clusters Liquid water 27Physical Chemistry Hydrophobic interactions Water is an excellent solvent for ions and for substances that contain polarized bonds (see p. 20). Substances of this type are referred to as polar or hydrophilic (“water-loving”). In contrast, substances that consist mainly of hydrocarbon structures dissolve only poorly in water. Such substances are said to be apolar or hydrophobic. A. Solubility of methane To understand the reasons for the poor water solubility of hydrocarbons, it is useful first to examine the energetics (see p.16) of the pro- cesses involved. In (1), the individual terms of the Gibbs–Helmholtz equation (see p. 20) for the simplest compound of this type, methane, are shown (see p. 4). As can be seen, the tran- sition from gaseous methane to water is ac- tually exothermic (∆H0 < 0). Nevertheless, the change in the free enthalpy ∆G0 is positive (the process is endergonic), because the en- tropy term T ∆S0 has a strongly positive value. The entropy change in the process (∆S0) is evidently negative—i. e., a solution of methane in water has a higher degree of order than either water or gaseous methane. One reason for this is that the methane molecules are less mobile when surrounded by water. More importantly, however, the water around the apolar molecules forms cage-like “clath- rate” structures, which—as in ice—are stabi- lized by H bonds. This strongly increases the degree of order in the water—and the more so the larger the area of surface contact between the water and the apolar phase. B. The “oil drop effect” The spontaneous separation of oil and water, a familiar observation in everyday life, is due to the energetically unfavorable formation of clathrate structures. When a mixture of water and oil is firmly shaken, lots of tiny oil drops form to begin with, but these quickly coalesce spontaneously to form larger drops—the two phases separate. A larger drop has a smaller surface area than several small drops with the same volume. Separation therefore reduces the area of surface contact between the water and the oil, and consequently also the extent of clathrate formation. The ∆S for this process is therefore positive (the disorder in the water increases), and the negative term –T ∆S makes the separation process exergonic (∆G < 0), so that it proceeds spontaneously. C. Arrangements of amphipathic substances in water Molecules that contain both polar and apolar groups are called amphipathic or amphiphilic. This group includes soaps (see p. 48), phos- pholipids (see p. 50), and bile acids (see p. 56). As a result of the “oil drop effect” amphi- pathic substances in water tend to arrange themselves in such a way as to minimize the area of surface contact between the apolar regions of the molecule and water. On water surfaces, they usually form single-layer films (top) in which the polar “head groups” face toward the water. Soap bubbles (right) consist of double films, with a thin layer of water enclosed between them. In water, depending on their concentration, amphipathic com- pounds form micelles—i. e., spherical aggre- gates with their head groups facing toward the outside, or extended bilayered double membranes. Most biological membranes are assembled according to this principle (see p. 214). Closed hollow membrane sacs are known as vesicles. This type of structure serves to transport substances within cells and in the blood (see p. 278). The separation of oil and water (B) can be prevented by adding a strongly amphipathic substance. During shaking, a more or less stable emulsion then forms, in which the sur- face of the oil drops is occupied by amphi- pathic molecules that provide it with polar properties externally. The emulsification of fats in food by bile acids and phospholipids is a vital precondition for the digestion of fats (see p. 314). 28 Basics 3 2 1 2 3 4 5 6 7 8 9 HB HB B OHBH % B 100 80 60 40 20 0 Cl H O H H O H H O H H O H H N H H H H O H H O H O H H H O H H NH H H Cl Gastric juice Lysosomes Sweat Urine Cytoplasm Blood plasma Small intestine A. Acids and bases Hydrogen chloride Very strong acid Water Very weak base Water Very weak acid Water Very weak base Water Very weak acid Ammonia Strong base Chloride ion Very weak base Hydronium ion Very strong acid Hydroxyl ion Very strong base Ammonium ion Weak acid Hydronium ion Very strong acid Hydroxyl ion Very strong base Proton exchange Proton exchange Proton exchange pKa = -7 pKa = 15.7 pKa = 9.2 Keq = 9 · 106 mol · l-1 Keq = 2 · 10-16 mol · l-1 Keq = 6 · 10-10 mol · l-1 C. BuffersB. pH values in the body pH Buffer solution: mixture of a weak acid with the conjugate base ∆ pH Base pKa Acid ∆ pH pH H2O H2O BaseAcid 31Physical Chemistry Redox processes A. Redox reactions Redox reactions are chemical changes in which electrons are transferred from one re- action partner to another (1; see also p.18). Like acid–base reactions (see p. 30), redox re- actions always involve pairs of compounds. A pair of this type is referred to as a redox system (2). The essential difference between the two components of a redox system is the number of electrons they contain. The more electronrich component is called the reduced form of the compound concerned, while the other one is referred to as the oxidized form. The reduced form of one system (the reducing agent) donates electrons to the oxidized form of another one (the oxidizing agent). In the process, the reducing agent becomes oxidized and the oxidizing agent is reduced (3). Any given reducing agent can reduce only certain other redox systems. On the basis of this type of observation, redox systems can be ar- ranged to form what are known as redox series (4). The position of a system within one of these series is established by its redox potential E (see p.18). The redox potential has a sign; it can be more negative or more positive than a reference potential arbitrarily set at zero (the normal potential of the system [2 H+/H2]). In addition, E depends on the con- centrations of the reactants and on the reac- tion conditions (see p.18). In redox series (4), the systems are arranged according to their increasing redox potentials. Spontaneous electron transfers are only possible if the re- dox potential of the donor is more negative than that of the acceptor (see p.18). B. Reduction equivalents In redox reactions, protons (H+) are often transferred along with electrons (e–), or pro- tons may be released. The combinations of electrons and protons that occur in redox processes are summed up in the term reduc- tion equivalents. For example, the combina- tion 1 e–/1 H+ corresponds to a hydrogen atom, while 2 e– and 2 H+ together produce a hydrogen molecule. However, this does not mean that atomic or molecular hydrogen is actually transferred from one molecule to the other (see below). Only the combination 2 e–/ 1 H+, the hydride ion, is transferred as a unit. C. Biological redox systems In the cell, redox reactions are catalyzed by enzymes, which work together with soluble or bound redox cofactors. Some of these factors contain metal ions as redox-active components. In these cases, it is usually single electrons that are transferred, with the metal ion changing its valency. Un- paired electrons often occur in this process, but these are located in d orbitals (see p. 2) and are therefore less dangerous than single electrons in non-metal atoms (“free radicals”; see below). We can only show here a few examples from the many organic redox systems that are found. In the complete reduction of the flavin coenzymes FMN and FAD (see p.104), 2 e– and 2 H+ are transferred. This occurs in two separate steps, with a semiquinone radi- cal appearing as an intermediate. Since or- ganic radicals of this type can cause damage to biomolecules, flavin coenzymes never oc- cur freely in solution, but remain firmly bound in the interior of proteins. In the reduction or oxidation of quinone/ quinol systems, free radicals also appear as intermediate steps, but these are less reactive than flavin radicals. Vitamin E, another qui- none-type redox system (see p.104), even functions as a radical scavenger, by delocaliz- ing unpaired electrons so effectively that they can no longer react with other molecules. The pyridine nucleotides NAD+ and NADP+ always function in unbound form. The oxi- dized forms contain an aromatic nicotinamide ring in which the positive charge is delocal- ized. The right-hand example of the two res- onance structures shown contains an elec- tron-poor, positively charged C atom at the para position to nitrogen. If a hydride ion is added at this point (see above), the reduced forms NADH or NADPH arise. No radical inter- mediate steps occur. Because a proton is re- leased at the same time, the reduced pyridine nucleotide coenzymes are correctly expressed as NAD(P)H+H+. 32 Basics Men+ Mem+ 2e2e 2e 2e O O H H H O H O H H N N N C NH CH3C H3C R O O N N N C NH CH3C H3C R O O H H N N N C NH CH3C H3C R O O H 2e 2H2e1H1e1e 1H e [H] H [H2] 1e 1H C C H R O H3CO H3CO O C C H R OH H3CO H3CO O OH C C H R OH H3CO H3CO e H e H e H e H e H O O O O H e H e H e H O H C N CONH H H H 2 H R H H N A N A P F A F e 1e 1H C N CONH H H H 2 H R C N CONH H H H 2 H R A. Redox reactions Redox system C Redox system B Electron exchange A red B ox A ox B red 1. Principle Redox system A 2. Redox systems Oxidizing agent Reducing agentbecomes reduced becomes oxidized 4. Redox series3. Possible electron 3. transfers Possible Not possible Transferred components Equivalent Electron Hydrogen atom Hydride ion Hydrogen molecule Metal complexes Oxidized Reduced Flavin Quinone/ hydro- quinone Reactive oxygen species (ROS) Oxidized flavin Semiquinone radical Reduced flavin p-Benzoquinone Semiquinone radical Hydroquinone Water Oxygen Hydroperoxyl radical Hydrogen peroxide Hydroxyl radical Water C. Biological redox systems NAD (P) NAD(P)H + H Hydride ionElectron-poor B. Reducing equivalents NAD(P) (Resonance structures) 33Physical Chemistry Chemistry of sugars A. Reactions of the monosaccharides The sugars (monosaccharides) occur in the metabolism in many forms (derivatives). Only a few important conversion reactions are discussed here, using D-glucose as an ex- ample. 1. Mutarotation. In the cyclic form, as op- posed to the open-chain form, aldoses have a chiral center at C-1 (see p. 34). The corre- sponding isomeric forms are called anomers. In the β-anomer (center left), the OH group at C-1 (the anomeric OH group) and the CH2OH group lie on the same side of the ring. In the α- anomer (right), they are on different sides. The reaction that interconverts anomers into each other is known as mutarotation (B). 2. Glycoside formation. When the anome- ric OH group of a sugar reacts with an alcohol, with elimination of water, it yields an O–glycoside (in the case shown, α –methylglu- coside). The glycosidic bond is not a normal ether bond, because the OH group at C-1 has a hemiacetal quality. Oligosaccharides and pol- ysaccharides also contain O-glycosidic bonds. Reaction of the anomeric OH group with an NH2 or NH group yields an N-glycoside (not shown). N-glycosidic bonds occur in nucleo- tides (see p. 80) and in glycoproteins (see p. 44), for example. 3. Reduction and oxidation. Reduction of the anomeric center at C-1 of glucose (2) pro- duces the sugar alcohol sorbitol. Oxidation of the aldehyde group at C-1 gives the intramo- lecular ester (lactone) of gluconic acid (a gly- conic acid). Phosphorylated gluconolactone is an intermediate of the pentose phosphate pathway (see p.152). When glucose is oxi- dized at C-6, glucuronic acid (a glycuronic acid) is formed. The strongly polar glucuronic acid plays an important role in biotransforma- tions in the liver (see pp.194, 316). 4. Epimerization. In weakly alkaline solu- tions, glucose is in equilibrium with the ketohexose D-fructose and the aldohexose D- mannose, via an enediol intermediate (not shown). The only difference between glucose and mannose is the configuration at C-2. Pairs of sugars of this type are referred to as epi- mers, and their interconversion is called epi- merization. 5. Esterification. The hydroxyl groups of monosaccharides can form esters with acids. In metabolism, phosphoric acid esters such as glucose 6-phosphate and glucose 1-phosphate (6) are particularly important. B. Polarimetry, mutarotation Sugar solutions can be analyzed by polarim- etry, a method based on the interaction be- tween chiral centers and linearly polarized light—i. e., light that oscillates in only one plane. It can be produced by passing normal light through a special filter (a polarizer). A second polarizing filter of the same type (the analyzer), placed behind the first, only lets the polarized light pass through when the polar- izer and the analyzer are in alignment. In this case, the field of view appears bright when one looks through the analyzer (1). Solutions of chiral substances rotate the plane of polar- ized light by an angle α either to the left or to the right. When a solution of this type is placed between the polarizer and the ana- lyzer, the field of view appears darker (2). The angle of rotation, α, is determined by turning the analyzer until the field of view becomes bright again (3). A solution’s optical rotation depends on the type of chiral com- pound, its concentration, and the thickness of the layer of the solution. This method makes it possible to determine the sugar content of wines, for example. Certain procedures make it possible to ob- tain the α and β anomers of glucose in pure form. A 1-molar solution of α-D-glucose has a rotation value [α]D of +112°, while a corre- sponding solution of β-D-glucose has a value of +19°. These values change spontaneously, however, and after a certain time reach the same end point of +52°. The reason for this is that, in solution, mutarotation leads to an equilibrium between the α and β forms in which, independently of the starting condi- tions, 62% of the molecules are present in the β form and 38% in the α form. 36 Biomolecules 1 6 1 23 6 α β 1 α 1 1 100 80 60 40 20 0 1. 2. 3. a a 10 20 4030 50 2 1 O HO OH OH H H H H COO OH H O HO OH OH H H H H O HOCH2 OH CH2OH HO OH OH H H H H HOCH2 O HO OH OH H H H H OH H CH2HO O HO OH OH H H H H H OH CH2HO O HO OH OH H H H H OH H CH2OPO O O O HO H H HO OH H H CH2OH OH H O HO OH H HO H H H H OH HOCH2 O HO OH OH H H H H H O CH3 HOCH2 A. Reactions of the monosaccharides B. Polarimetry, mutarotation Polarizer Analyzer α (˚) 62% β 38% α α-D-Glucose: [ α ] D = +112° β-D-Glucose: [α] D = +19° Time (min) Su ga r W at er Su ga r β-D-Glucose α-D-Glucose Mutarotation Esterification α-Methyl- glucoside Glycoside formation D-FructoseGlucose 6- phosphate α-D-Mannose Epimerization SorbitolGlucuronate Gluconolactone Oxidation ReductionOxidation + 52° 37Carbohydrates Monosaccharides and disaccharides A. Important monosaccharides Only the most important of the large number of naturally occurring monosaccharides are mentioned here. They are classified according to the number of C atoms (into pentoses, hexoses, etc.) and according to the chemical nature of the carbonyl function into aldoses and ketoses. The best-known aldopentose (1), D-ribose, is a component of RNA and of nucleotide coenzymes and is widely distributed. In these compounds, ribose always exists in the fura- nose form (see p. 34). Like ribose, D-xylose and L-arabinose are rarely found in free form. However, large amounts of both sugars are found as constituents of polysaccharides in the walls of plant cells (see p. 42). The most important of the aldohexoses (1) is D-glucose. A substantial proportion of the biomass is accounted for by glucose polymers, above all cellulose and starch. Free D-glucose is found in plant juices (“grape sugar”) and as “blood sugar” in the blood of higher animals. As a constituent of lactose (milk sugar), D- galactose is part of the human diet. Together with D-mannose, galactose is also found in glycolipids and glycoproteins (see p. 44). Phosphoric acid esters of the ketopentose D-ribulose (2) are intermediates in the pen- tose phosphate pathway (see p.152) and in photosynthesis (see p.128). The most widely distributed of the ketohexoses is D-fructose. In free form, it is present in fruit juices and in honey. Bound fructose is found in sucrose (B) and plant polysaccharides (e. g., inulin). In the deoxyaldoses (3), an OH group is replaced by a hydrogen atom. In addition to 2-deoxy-D-ribose, a component of DNA (see p. 84) that is reduced at C-2, L-fucose is shown as another example of these. Fucose, a sugar in the λ series (see p. 34) is reduced at C-6. The acetylated amino sugars N-acetyl-D- glucosamine and N-acetyl-D-Galactosamine (4) are often encountered as components of glycoproteins. N-acetylneuraminic acid (sialic acid, 5), is a characteristic component of glycoproteins. Other acidic monosaccharides such as D-glu- curonic acid, D-galacturonic acid, and liduronic acid, are typical constituents of the glycosa- minoglycans found in connective tissue. Sugar alcohols (6) such as sorbitol and mannitol do not play an important role in animal metabolism. B. Disaccharides When the anomeric hydroxyl group of one monosaccharide is bound glycosidically with one of the OH groups of another, a disaccha- ride is formed. As in all glycosides, the glyco- sidic bond does not allow mutarotation. Since this type of bond is formed stereospecifically by enzymes in natural disaccharides, they are only found in one of the possible configura- tions (α or β). Maltose (1) occurs as a breakdown product of the starches contained in malt (“malt sugar”; see p.148) and as an intermediate in intestinal digestion. In maltose, the anomeric OH group of one glucose molecule has an α- glycosidic bond with C-4 in a second glucose residue. Lactose (“milk sugar,” 2) is the most impor- tant carbohydrate in the milk of mammals. Cow’s milk contains 4.5% lactose, while hu- man milk contains up to 7.5%. In lactose, the anomeric OH group of galactose forms a β- glycosidic bond with C-4 of a glucose. The lactose molecule is consequently elongated, and both of its pyran rings lie in the same plane. Sucrose (3) serves in plants as the form in which carbohydrates are transported, and as a soluble carbohydrate reserve. Humans value it because of its intensely sweet taste. Sources used for sucrose are plants that contain par- ticularly high amounts of it, such as sugar cane and sugar beet (cane sugar, beet sugar). Enzymatic hydrolysis of sucrose-containing flower nectar in the digestive tract of bees— catalyzed by the enzyme invertase—produces honey, a mixture of glucose and fructose. In sucrose, the two anomeric OH groups of glu- cose and fructose have a glycosidic bond; su- crose is therefore one of the non-reducing sugars. 38 Biomolecules O OH HO H H H H O O OH OH H H H H H O H O CH2 O OH OH H H H H O O OH OH H H H H H O H O OH OH H H H H O OH OH H H H H H OO OH HO HO HO CH2 CH2 CH2 HO HOCH2 CH2 O NHCOCH3 H H H H O OH NHCOCH3 H H H H H O NHCOCH3 H H H H O OH NHCOCH3 H H H H HH O O O O O H O O C CH3C C O NH C O NH H3C HO HOCH2 CH2 HO HOCH2 CH2 H H 1 4 6 α β 1 4 α 1 4 α 1 4 α 1 α 1 4 β 1 4 β 1 4 23 Glycogen – branched homopolymer Mono- saccharide 1 D-GlcNAc D-Glc D-Gal D-Gal D-Glc D-Glc L-Ara D-Glc D-Glc D-Fru D-GlcNAc D-Glc D-GlcUA Occurrence Cell wall Slime Red algae (agar) Red algae Cell wall Cell wall (Hemicellulose) Cell wall (pectin) Amyloplasts Amyloplasts Storage cells Insects, crabs Liver, muscle Connective tissue Mono- saccharide 2 D-MurNAc1) L-aGal2) D-Xyl (D-Gal, L-Fuc) Function SC WB WB WB SC SC SC RC RC RC SK RK SK,WB 1 3 1 3 1 4 1 6 1 2) 1 3 1 6 1 6 SC= structural carbohydrate, RC= reserve carbohydrate, WB = water-binding carbohydrate; 1) N-acetylmuramic acid, 2) 3,6-anhydrogalactose Linkage Branch- ing β α β β β β α α α β β α β β α β α β β α α α Poly- saccharide Bacteria Murein Dextran Plants Agarose Carrageenan Cellulose Xyloglucan Arabinan Amylose Amylopectin Inulin Animals Chitin Glycogen Hyaluronic acid A. Polysaccharides: structure B. Important polysaccharides Reducing end Peptide D-GlcNAc 1 4 1 6 1 4 1 3 1 4 1 4 1 5 1 4 1 4 2 1 1 4 1 4 1 4 1 3 ( Murein – linear heteropolymer 41Carbohydrates Plant polysaccharides Two glucose polymers of plant origin are of special importance among the polysac- charides: β14-linked polymer cellulose and starch, which is mostly α14-linked. A. Cellulose Cellulose, a linear homoglycan of β14- linked glucose residues, is the most abundant organic substance in nature. Almost half of the total biomass consists of cellulose. Some 40–50% of plant cell walls are formed by cel- lulose. The proportion of cellulose in cotton fibers, an important raw material, is 98%. Cel- lulose molecules can contain more than 104 glucose residues (mass 1–2 106 Da) and can reach lengths of 6–8 µm. Naturally occurring cellulose is extremely mechanically stable and is highly resistant to chemical and enzymatic hydrolysis. These properties are due to the conformation of the molecules and their supramolecular or- ganization. The unbranched β14 linkage re- sults in linear chains that are stabilized by hydrogen bonds within the chain and be- tween neighboring chains (1). Already during biosynthesis, 50–100 cellulose molecules as- sociate to form an elementary fibril with a diameter of 4 nm. About 20 such elementary fibrils then form a microfibril (2), which is readily visible with the electron microscope. Cellulose microfibrils make up the basic framework of the primary wall of young plant cells (3), where they form a complex network with other polysaccharides. The linking poly- saccharides include hemicellulose, which is a mixture of predominantly neutral heterogly- cans (xylans, xyloglucans, arabinogalactans, etc.). Hemicellulose associates with the cellu- lose fibrils via noncovalent interactions. These complexes are connected by neutral and acidic pectins, which typically contain galac- turonic acid. Finally, a collagen-related protein, extensin, is also involved in the for- mation of primary walls. In the higher animals, including humans, cellulose is indigestible, but important as roughage (see p. 273). Many herbivores (e. g., the ruminants) have symbiotic unicellular or- ganisms in their digestive tracts that break down cellulose and make it digestible by the host. B. Starch Starch, a reserve polysaccharide widely dis- tributed in plants, is the most important car- bohydrate in the human diet. In plants, starch is present in the chloroplasts in leaves, as well as in fruits, seeds, and tubers. The starch con- tent is especially high in cereal grains (up to 75% of the dry weight), potato tubers (ap- proximately 65%), and in other plant storage organs. In these plant organs, starch is present in the form of microscopically small granules in special organelles known as amyloplasts. Starch granules are virtually insoluble in cold water, but swell dramatically when the water is heated. Some 15–25% of the starch goes into solution in colloidal form when the mix- ture is subjected to prolonged boiling. This proportion is called amylose (“soluble starch”). Amylose consists of unbranched α14- linked chains of 200–300 glucose residues. Due the α configuration at C-1, these chains form a helix with 6–8 residues per turn (1). The blue coloring that soluble starch takes on when iodine is added (the “iodine–starch re- action”) is caused by the presence of these helices—the iodine atoms form chains inside the amylose helix, and in this largely non- aqueous environment take on a deep blue color. Highly branched polysaccharides turn brown or reddishbrown in the presence of iodine. Unlike amylose, amylopectin, which is practically insoluble, is branched. On average, one in 20–25 glucose residues is linked to another chain via an α16 bond. This leads to an extended tree-like structure, which— like amylose—contains only one anomeric OH group (a “reducing end”). Amylopectin molecules can contain hundreds of thousands of glucose residues; their mass can be more than 108 Da. 42 Biomolecules α 1 1 α 4 6 HO HO OH O O O O OO O O O O O O OO O OH OH OH OH O O O HO O O α 1 4 1. 2. 3. Ca 2 β 1 4 23 5 6 3 12 4 5 6 Ca 2 A. Cellulose B. Starch Microfibril Elementary fibril 2. Amylopectin 80%1. Amylose 20% Pectin Hemi- cellulose Cellulose microfibrilExtensin Reducing end Plant cell Endoplasmic reticulum Mitochondria Golgi apparatus Nucleus Vacuole Chloroplasts StarchStarch Plasma membrane 43Carbohydrates Overview A. Classification The lipids are a large and heterogeneous group of substances of biological origin that are easily dissolved in organic solvents such as methanol, acetone, chloroform, and ben- zene. By contrast, they are either insoluble or only poorly soluble in water. Their low water solubility is due to a lack of polarizing atoms such as O, N, S, and P (see p. 6). Lipids can be classified into substances that are either hydrolyzable— i. e., able to undergo hydrolytic cleavage—or nonhydrolyzable. Only a few examples of the many lipids known can be mentioned here. The individual classes of lipids are discussed in more detail in the fol- lowing pages. Hydrolyzable lipids (components shown in brackets). The simple esters include the fats (triacylglycerol; one glycerol + three acyl res- idues); the waxes (one fatty alcohol + one acyl residue); and the sterol esters (one sterol + one acyl residue). The phospholipids are esters with more complex structures. Their charac- teristic component is a phosphate residue. The phospholipids include the phosphatidic acids (one glycerol + two acyl residues + one phosphate) and the phosphatides (one glyc- erol + two acyl residues + one phosphate + one amino alcohol). In the sphingolipids, glyc- erol and one acyl residue are replaced by sphingosine. Particularly important in this group are the sugar-containing glycolipids (one sphingosine + one fatty acid + sugar). The cerebrosides (one sphingosine + one fatty acid + one sugar) and gangliosides (one sphin- gosine + one fatty acid + several different sugars, including neuraminic acid) are repre- sentatives of this group. The components of the hydrolyzable lipids are linked to one another by ester bonds. They are easily broken down either enzymatically or chemically. Non-hydrolyzable lipids. The hydrocarbons include the alkanes and carotenoids. The lipid alcohols are also not hydrolyzable. They in- clude long-chained alkanols and cyclic sterols such as cholesterol, and steroids such as es- tradiol and testosterone. The most important acids among the lipids are fatty acids. The eicosanoids also belong to this group; these are derivatives of the polyunsaturated fatty acid arachidonic acid (see p. 390). B. Biological roles 1. Fuel. Lipids are an important source of en- ergy in the diet. In quantitative terms, they represent the principal energy reserve in ani- mals. Neutral fats in particular are stored in specialized cells, known as adipocytes. Fatty acids are released from these again as needed, and these are then oxidized in the mitochon- dria to form water and carbon dioxide, with oxygen being consumed. This process also gives rise to reduced coenzymes, which are used for ATP production in the respiratory chain (see p.140). 2. Nutrients. Amphipathic lipids are used by cells to build membranes (see p. 214). Typ- ical membrane lipids include phospholipids, glycolipids, and cholesterol. Fats are only weakly amphiphilic and are therefore not suitable as membrane components. 3. Insulation. Lipids are excellent insula- tors. In the higher animals, neutral fats are found in the subcutaneous tissue and around various organs, where they serve as mechan- ical and thermal insulators. As the principal constituent of cell membranes, lipids also in- sulate cells from their environment mechan- ically and electrically. The impermeability of lipid membranes to ions allows the formation of the membrane potential (see p.126). 4. Special tasks. Some lipids have adopted special roles in the body. Steroids, eicosa- noids, and some metabolites of phospholipids have signaling functions. They serve as hor- mones, mediators, and second messengers (see p. 370). Other lipids form anchors to at- tach proteins to membranes (see p. 214). The lipids also produce cofactors for enzymatic re- actions—e. g., vitamin K (see p. 52) and ubiq- uinone (see p.104). The carotenoid retinal, a light-sensitive lipid, is of central importance in the process of vision (see p. 358). Several lipids are not formed indepen- dently in the human body. These substances, as essential fatty acids and fat-soluble vita- mins, are indispensable components of nutri- tion (see pp. 364ff.) 46 Biomolecules O2 CO2 H2O C O O CH2 C O O C O CH2 O C O CH O H2C O P O O O N CH3 CH2 CH3 C O CH2 O C O CH O C O CH2 O HO OH COOH C NH O OH OH O OH OH O CH2 HO HO C O CH2 O C O CH O H2C O P O O O 4. Special tasks 1. Fuel 2. Building block 3. Thermal insulator Fat Glycerol Fatty acid Mitochondrion 37 °C 0 °C Phospho- lipid Membrane Lipid bilayer Cytoplasm Cell Signaling Cofactor Visual pigment Anchor A. Classification B. Biological roles Hydrolyzable lipids Non-hydrolyzable lipids Hydrocarbons Alkanes Carotenoids Esters Fats Waxes Sterol esters Phospholipids Phosphatidates Phosphatids Sphingolipids Alcohols Long-chain alkanols Sterols Steroids Glycolipids Cerebrosides Gangliosides Acids Fatty acids Eicosanoids ADP+Pi ATP CoQ 47Lipids Fatty acids and fats A. Carboxylic acids The naturally occurring fatty acids are carbox- ylic acids with unbranched hydrocarbon chains of 4–24 carbon atoms. They are present in all organisms as components of fats and membrane lipids. In these com- pounds, they are esterified with alcohols (glycerol, sphingosine, or cholesterol). How- ever, fatty acids are also found in small amounts in unesterified form. In this case, they are known as free fatty acids (FFAs). As free fatty acids have strongly amphipathic properties (see p. 28), they are usually present in protein-bound forms. The table lists the full series of aliphatic carboxylic acids that are found in plants and animals. In higher plants and animals, un- branched, longchain fatty acids with either 16 or 18 carbon atoms are the most common— e. g., palmitic and stearic acid. The number of carbon atoms in the longer, natural fatty acids is always even. This is because they are bio- synthesized from C2 building blocks (see p.168). Some fatty acids contain one or more isolated double bonds, and are therefore “un- saturated.” Common unsaturated fatty acids include oleic acid and linoleic acid. Of the two possible cis–trans isomers (see p. 8), usually only the cis forms are found in natural lipids. Branched fatty acids only occur in bacteria. A shorthand notation with several numbers is used for precise characterization of the struc- ture of fatty acids—e g., 18:2;9,12 for linoleic acid. The first figure stands for the number of C atoms, while the second gives the number of double bonds. The positions of the double bonds follow after the semicolon. As usual, numbering starts at the carbon with the high- est oxidation state (i. e., the carboxyl group corresponds to C-1). Greek letters are also commonly used (α = C-2; β = C-3; ω = the last carbon, ω-3 = the third last carbon). Essential fatty acids are fatty acids that have to be supplied in the diet. Without ex- ception, these are all polyunsaturated fatty acids: the C20 fatty acid arachidonic acid (20:4;5,8,11,14) and the two C18 acids linoleic acid (18:2;9,12) and linolenic acid (18:3;9,12,15). The animal organism requires arachidonic acid to synthesize eicosanoids (see p. 390). As the organism is capable of elongating fatty acids by adding C2 units, but is not able to introduce double bonds into the end sections of fatty acids (after C-9), arachi- donic acid has to be supplied with the diet. Linoleic and linolenic acid can be converted into arachidonic acid by elongation, and they can therefore replace arachidonic acid in the diet. B. Structure of fats Fats are esters of the trivalent alcohol glycerol with three fatty acids. When a single fatty acid is esterified with glycerol, the product is re- ferred to as a monoacylglycerol (fatty acid res- idue = acyl residue). Formally, esterification with additional fatty acids leads to diacylglycerol and ulti- mately to triacylglycerol, the actual fat (for- merly termed “triglyceride”). As triacylglycer- ols are uncharged, they are also referred to as neutral fats. The carbon atoms of glycerol are not usually equivalent in fats. They are distin- guished by their “sn” number, where sn stands for “stereospecific numbering.” The three acyl residues of a fat molecule may differ in terms of their chain length and the number of double bonds they contain. This results in a large number of possible combinations of individual fat molecules. When extracted from biological materials, fats always represent mixtures of very similar compounds, which differ in their fatty acid residues. A chiral center can arise at the mid- dle C atom (sn -C-2) of a triacylglycerol if the two external fatty acids are different. The monoacylglycerols and diacylglycerols shown here are also chiral compounds. Nutritional fats contain palmitic, stearic, oleic acid, and linoleic acid particularly often. Unsaturated fatty acids are usually found at the central C atom of glycerol. The length of the fatty acid residues and the number of their double bonds affect the melting point of the fats. The shorter the fatty acid residues and the more double bonds they contain, the lower their melting points. 48 Biomolecules SO3 GalGalNAcGalGlc NeuAc P P P 2 P P C O CH2 O C O C H2C O P O O O O (CH2)2 H N CH3 CH3 CH3 N CH3 CH3 CH3 CH2CH2HO CH2HO CH NH3 COO CH2HO CH2 NH3 HO OH OH OH OH OH H H H H H H C O NH CH C CH2 C OH H C H H O P O O O (CH2)2 N CH3 CH3 CH3 A. Structure of fats, phospholipids, and glycolipids Acyl residue 1 G ly ce ro l 1. Fats Fat Phosphatidate Phosphatide (phosphatidylcholine, lecithin) Amino alcohol or sugar alcoholPhosphatide Choline Serine myo-InositolEthanolamine Acyl residue 2 Acyl residue 3 Acyl residue 1 Acyl residue 2 Sphingosine Sphingosine Amino alcohol or sugar alcohol Lysophospholipid G ly ce ro l G ly ce ro l Sphingophospholipid Amino alcohol or sugar alcohol Acyl residue 1Acyl residue 1 Acyl residue 1 G ly ce ro l Sphingosine Sphingosine Sugar Sphingosine 2. Phospholipids Ceramide Sphingomyelin Sphingosine Sugar Sulfatide Sphingosine Choline Acyl residue 1 Acyl residue 1 Acyl residue 1 Acyl residue 1 3. Sphingolipids Cerebroside (galactosyl or glycosyl ceramide) Ganglioside GM1 Acyl residue 1 Acyl residue 2 51Lipids Isoprenoids A. Activated acetic acid as a component of lipids Although the lipids found in plant and animal organisms occur in many different forms, they are all closely related biogenetically; they are all derived from acetyl-CoA, the “ac- tivated acetic acid” (see pp.12, 110). 1. One major pathway leads from acetyl- CoA to the activated fatty acids (acyl-CoA; for details, see p.168). Fats, phospholipids, and glycolipids are synthesized from these, and fatty acid derivatives in particular are formed. Quantitatively, this is the most important pathway in animals and most plants. 2. The second pathway leads from acetyl- CoA to isopentenyl diphosphate (“active iso- prene”), the basic component for the isopren- oids. Its biosynthesis is discussed in connec- tion with biosynthesis of the isoprenoid, cho- lesterol (see p.172). B. Isoprenoids Formally, isoprenoids are derived from a sin- gle common building block, isoprene (2- methyl-1,3-butadiene), a methyl-branched compound with five C atoms. Activated isoprene, isopentenyl diphosphate, is used by plants and animals to biosynthesize linear and cyclic oligomers and polymers. For the isoprenoids listed here—which only represent a small selection—the number of isoprene units (I) is shown. From activated isoprene, the metabolic pathway leads via dimerization to activated geraniol (I = 2) and then to activated farnesol (I = 3). At this point, the pathway divides into two. Further extension of farnesol leads to chains with increasing numbers of isoprene units—e. g., phytol (I = 4), dolichol (I = 14–24), and rubber (I = 700–5000). The other pathway involves a “head-to-head” linkage between two farnesol residues, giving rise to squalene (I = 6), which, in turn, is converted to choles- terol (I = 6) and the other steroids. The ability to synthesize particular iso- prenoids is limited to a few species of plants and animals. For example, rubber is only formed by a few plant species, including the rubber tree (Hevea brasiliensis). Several iso- prenoids that are required by animals for me- tabolism, but cannot be produced by them independently, are vitamins; this group includes vitamins A, D, E, and K. Due to its structure and function, vitamin D is now usu- ally classified as a steroid hormone (see pp. 56, 330). Isoprene metabolism in plants is very com- plex. Plants can synthesize many types of ar- omatic substances and volatile oils from iso- prenoids. Examples include menthol (I= 2 ), camphor (I = 2), and citronellal (I = 2). These C10 compounds are also called monoterpenes. Similarly, compounds consisting of three iso- prene units (I = 3) are termed sesquiterpenes, and the steroids (I = 6) are called triterpenes. Isoprenoids that have hormonal and sig- naling functions form an important group. These include steroid hormones (I = 6) and retinoate (the anion of retinoic acid; I = 3) in vertebrates, and juvenile hormone (I = 3) in arthropods. Some plant hormones also belong to the isoprenoids—e. g., the cytokinins, absci- sic acid, and brassinosteroids. Isoprene chains are sometimes used as lipid anchors to fix molecules to membranes (see p. 214). Chlorophyll has a phytyl residue (I = 4) as a lipid anchor. Coenzymes with iso- prenoid anchors of various lengths include ubiquinone (coenzyme Q; I = 6–10), plastoqui- none (I = 9), and menaquinone (vitamin K; I = 4–6). Proteins can also be anchored to mem- branes by isoprenylation. In some cases, an isoprene residue is used as an element to modify molecules chemi- cally. One example of this is N'-isopentenyl- AMP, which occurs as a modified component in tRNA. 52 Biomolecules O CH3C A S O C A S O P P 2 1 O CH3 H3C CH3 CH2OH HO CH2OH CH2OH O CH3 H3C HO CH3 CH3 O O CH3 O CH3 O CH3 n = 6–10 OH CH3 OH A. Activated acetic acid as a component of lipids Active isopreneActivated fatty acid Activated acetic acid Acetyl-CoA Acyl-CoA Isopentenyl diphosphate Fats Phospholipids Glycolipids Isoprenoids Isoprene Metabolite modified with isoprene Active isoprene I =1 Isopentenyl-AMP I =1 Camphor I = 2 Menthol I = 2 Cholesterol I = 6 Citronellol I = 2 Juvenile hormone I = 3 Geraniol I = 2 Farnesol I = 3 Squalene I = 6 Chain-like isoprenoids Building block of all isoprenoids Biosynthesis only in plants and micro-organisms B. Isoprenoids Steroid hormones Bile acids Steroid glycosides I = 6 Phytol I = 4 Dolichol I = 14 – 24 Rubber I = 700 – 5 000 Menaquinone I = 4 – 6 Plastoquinone I = 9 Retinoic Carotenoids (Vitamin A) Tocopherol (Vitamin E) I = 4 Phylloquinone (Vitamin K) I = 4 Cyclic isoprenoids Ubiquinone I = 6 – 10 53Lipids Steroids: overview The three most important groups of steroids are the sterols, bile acids, and steroid hor- mones. Particularly in plants, compounds with steroid structures are also found that are notable for their pharmacological ef- fects—steroid alkaloids, digitalis glycosides, and saponins. A. Sterols Sterols are steroid alcohols. They have a β-positioned hydroxyl group at C-3 and one or more double bonds in ring B and in the side chain. There are no further oxygen functions, as in the carbonyl and carboxyl groups. The most important sterol in animals is cholesterol. Plants and microorganisms have a wide variety of closely related sterols in- stead of cholesterol—e. g., ergosterol, β-sitos- terol, and stigmasterol. Cholesterol is present in all animal tissues, and particularly in neural tissue. It is a major constituent of cellular membranes, in which it regulates fluidity (see p. 216). The storage and transport forms of cholesterol are its esters with fatty acids. In lipoproteins, cholesterol and its fatty acid esters are associated with other lipids (see p. 278). Cholesterol is a con- stituent of the bile and is therefore found in many gallstones. Its biosynthesis, metabo- lism, and transport are discussed elsewhere (see pp.172, 312). Cholesterol-rich lipoproteins of the LDL type are particularly important in the devel- opment of arteriosclerosis, in which the arte- rial walls are altered in connection with an excess plasma cholesterol level. In terms of dietary physiology, it is important that plant foodstuffs are low in cholesterol. By contrast, animal foods can contain large amounts of cholesterol—particularly butter, egg yolk, meat, liver, and brain. B. Bile acids Bile acids are synthesized from cholesterol in the liver (see p. 314). Their structures can therefore be derived from that of cholesterol. Characteristic for the bile acids is a side chain shortened by three C atoms in which the last carbon atom is oxidized to a carboxyl group. The double bond in ring B is reduced and rings A and B are in cis position relative to each other (see p. 54). One to three hydroxyl groups (in α position) are found in the steroid core at positions 3, 7, and 12. Bile acids keep bile cholesterol in a soluble state as micelles and promote the digestion of lipids in the intestine (see p. 270). Cholic acid and cheno- deoxycholic acid are primary bile acids that are formed by the liver. Their dehydroxylation at C-7 by microorganisms from the intestinal flora gives rise to the secondary bile acids lithocholic acid and deoxycholic acid. C. Steroid hormones The conversion of cholesterol to steroid hormones (see p. 376) is of minor importance quantitatively, but of major importance in terms of physiology. The steroid hormones are a group of lipophilic signal substances that regulate metabolism, growth, and repro- duction (see p. 374). Humans have six steroid hormones: progesterone, cortisol, aldosterone, testos- terone, estradiol, and calcitriol. With the ex- ception of calcitriol, these steroids have either no side chain or only a short side one consist- ing of two carbons. Characteristic for most of them is an oxo group at C-3, conjugated with a double bond between C-4 and C-5 of ring A. Differences occur in rings C and D. Estradiol is aromatic in ring A, and its hydroxyl group at C-3 is therefore phenolic. Calcitriol differs from other vertebrate steroid hormones; it still contains the complete carbon framework of cholesterol, but lightdependent opening of ring B turns it into what is termed a “secoste- roid” (a steroid with an open ring). Ecdysone is the steroid hormone of the arthropods. It can be regarded as an early form of the steroid hormones. Steroid hor- mones with signaling functions also occur in plants. 56 Biomolecules HO HO HO OH HO OH OH O OH HO O HO OH OH O OH HO O OH O HO C OH CH2OH O OHC O HO C CH2OH O O OH O C CH3 O OH OH CH2 HO 13 25 OH OH HO HO O OH HO H HO OH C. Steroid hormones Molting hormone of insects, spiders and crabs Cortisol Aldosterone Testosterone Estradiol Progesterone Calcitriol Ecdysone Cholic acid Lithocholic acid Cheno- deoxycholic acid Deoxy- cholic acid Cholesterol Ergosterol Stigmasterol Animal sterol β-Sitosterol Plant sterols A. Sterols B. Bile acids 57Lipids Amino acids: chemistry and properties A. Amino acids: functions The amino acids (2-aminocarboxylic acids) fulfill various functions in the organism. Above all, they serve as the components of peptides and proteins. Only the 20 proteino- genic amino acids (see p. 60) are included in the genetic code and therefore regularly found in proteins. Some of these amino acids undergo further (post-translational) change following their incorporation into proteins (see p. 62). Amino acids or their derivatives are also form components of lipids—e. g., ser- ine in phospholipids and glycine in bile salts. Several amino acids function as neurotransmitters themselves (see p. 352), while others are precursors of neurotransmit- ters, mediators, or hormones (see p. 380). Amino acids are important (and sometimes essential) components of food (see p. 360). Specific amino acids form precursors for other metabolites—e. g., for glucose in gluconeogen- esis, for purine and pyrimidine bases, for heme, and for other molecules. Several non- proteinogenic amino acids function as inter- mediates in the synthesis and breakdown of proteinogenic amino acids (see p. 412) and in the urea cycle (see p.182). B. Optical activity The natural amino acids are mainly α-amino acids, in contrast to β-amino acids such as β- alanine and taurine. Most α-amino acids have four different substituents at C-2 (Cα). The α atom therefore represents a chiral center—i. e., there are two different enantiomers (L- and D-amino acids; see p. 8). Among the proteino- genic amino acids, only glycine is not chiral (R = H). In nature, it is almost exclusively L-amino acids that are found. D-Amino acids occur in bacteria—e. g., in murein (see p. 40)—and in peptide antibiotics. In animal metabolism, D-Amino acids would disturb the enzymatic reactions of L-amino acids and they are therefore broken down in the liver by the enzyme D-amino acid oxidase. The Fischer projection (center) is used to present the formulas for chiral centers in bio- molecules. It is derived from their three-di- mensional structure as follows: firstly, the tetrahedron is rotated in such a way that the most oxidized group (the carboxylate group) is at the top. Rotation is then continued until the line connecting line COO– and R (red) is level with the page. In L-amino acids, the NH3 + group is then on the left, while in D- amino acids it is on the right. C. Dissociation curve of histidine All amino acids have at least two ionizable groups, and their net charge therefore de- pends on the pH value. The COOH groups at the α-C atom have pKa values of between 1.8 and 2.8 and are therefore more acidic than simple monocarboxylic acids. The basicity of the α-amino function also varies, with pKa values of between 8.8 and 10.6, depending on the amino acid. Acidic and basic amino acids have additional ionizable groups in their side chain. The pKa values of these side chains are listed on p. 60. The electrical charges of peptides and proteins are mainly determined by groups in the side chains, as most α-car- boxyl and α-amino functions are linked to peptide bonds (see p. 66). Histidine can be used here as an example of the pH-dependence of the net charge of an amino acid. In addition to the carboxyl group and the amino group at the α-C atom with pKa values of 1.8 and 9.2, respectively, histidine also has an imidazole residue in its side chain with a pKa value of 6.0. As the pH increases, the net charge (the sum of the positive and negative charges) therefore changes from +2 to –1. At pH 7.6, the net charge is zero, even though the molecule contains two almost completely ionized groups in these condi- tions. This pH value is called the isoelectric point. At its isoelectric point, histidine is said to be zwitterionic, as it has both anionic and cationic properties. Most other amino acids are also zwitterionic at neutral pH. Peptides and proteins also have isoelectric points, which can vary widely depending on the composition of the amino acids. 58 Biomolecules –2.4 –1.9 –2.0 –2.3 –2.2 –1.2 –1.5 8.3 +0.8 +6.1 +5.9 +6.0 +5.1 +4.9 10.1 6.04.3 4.0 12.5 10.8 +9.7 +9.4 +11.0 +10.2 +10.3 +15.0 +20.0 H CH3 CH CH3 H3C CH2 CH CH3 H3C C CH2 H3C H CH3 CH2 SH CH2 CH2 S CH3 C OH H3C HCH2 OHCH HN H2C CH2 CH2 COO N H CH2CH2 OH CH2 CH2 CONH2 CH2 CH2 CONH2 CH2 COO CH2 HN HC N CH CH2 CH2 CH2 NH C H2N NH2 CH2 CH2 CH2 CH2 NH3 CH2 CH2 COO (Gly, G) (Ala, A) (Val, V) (Leu, L) (Ile, I) (Cys, C) (Met, M) Glycine Cysteine Aliphatic Alanine Valine Leucine Isoleucine Methionine Sulfur-containing pKa value A. The proteinogenic amino acids Chiral center (Phe, F) (Tyr, Y) (Trp, W) (Pro, P) (Ser, S) (Thr, T) Proline Aromatic Cyclic Phenylalanine Tyrosine Tryptophan Serine Threonine Neutral (Asn,N) (Gln, Q) (Asp, D) (Glu, E) (His, H) (Lys,K) (Arg, R) Aspartic acid Acidic Asparagine Glutamine Glutamic acid Histidine Lysine Arginine Neutral Basic Polarity Essential amino acids Indole ring Pyrrolidine ring Imidazole ring 61Amino Acids Non-proteinogenic amino acids In addition to the 20 proteinogenic amino acids (see p. 60), there are also many more compounds of the same type in nature. These arise during metabolic reactions (A) or as a result of enzymatic modifications of amino acid residues in peptides or proteins (B). The “biogenic amines” (C) are synthesized from α- amino acids by decarboxylation. A. Rare amino acids Only a few important representatives of the non-proteinogenic amino acids are men- tioned here. The basic amino acid ornithine is an analogue of lysine with a shortened side chain. Transfer of a carbamoyl residue to or- nithine yields citrulline. Both of these amino acids are intermediates in the urea cycle (see p.182). Dopa (an acronym of 3,4-dihydroxy- phenylalanine) is synthesized by hydroxyla- tion of tyrosine. It is an intermediate in the biosynthesis of catecholamines (see p. 352) and of melanin. It is in clinical use in the treatment of Parkinson’s disease. Selenocys- teine, a cysteine analogue, occurs as a compo- nent of a few proteins—e. g., in the enzyme glutathione peroxidase (see p. 284). B. Post-translational protein modification Subsequent alteration of amino acid residues in finished peptides and proteins is referred to as post-translational modification. These re- actions usually only involve polar amino acid residues, and they serve various purposes. The free α-amino group at the N-terminus is blocked in many proteins by an acetyl res- idue or a longer acyl residue (acylation). N- terminal glutamate can cyclize into a pyroglu- tamate residue, while the C-terminal carbox- ylate group can be present in an amidated form (see TSH, p. 380). The side chains of ser- ine and asparagine residues are often linked to oligosaccharides (glycosylation, see p. 230). Phosphorylation of proteins mainly affects serine and tyrosine residues. These reactions have mainly regulatory functions (see p.114). Aspartate and histidine residues of enzymes are sometimes phosphorylated, too. A special modification of glutamate residues, -carbox- ylation, is found in coagulation factors. It is essential for blood coagulation (see p. 290). The ε-amino group of lysine residues is sub- ject to a particularly large number of modifi- cations. Its acetylation (or deacetylation) is an important mechanism for controlling genetic activity (see p. 244). Many coenzymes and cofactors are covalently linked to lysine resi- dues. These include biotin (see p.108), lipoic acid (see p.106), and pyridoxal phosphate (see p.108), as well as retinal (see p. 358). Covalent modification with ubiquitin marks proteins for breakdown (see p.176). In colla- gen, lysine and proline residues are modified by hydroxylation to prepare for the formation of stable fibrils (see p. 70). Cysteine residues form disulfide bonds with one another (see p. 72). Cysteine prenylation serves to anchor proteins in membranes (see p. 214). Covalent bonding of a cysteine residue with heme oc- curs in cytochrome c. Flavins are sometimes covalently bound to cysteine or histidine res- idues of enzymes. Among the modifications of tyrosine residues, conversion into iodinated thyroxine (see p. 374) is particularly interest- ing. C. Biogenic amines Several amino acids are broken down by de- carboxylation. This reaction gives rise to what are known as biogenic amines, which have various functions. Some of them are compo- nents of biomolecules, such as ethanolamine in phospholipids (see p. 50). Cysteamine and -alanine are components of coenzyme A (see p.12) and of pantetheine (see pp.108, 168). Other amines function as signaling substan- ces. An important neurotransmitter derived from glutamate is γ-aminobutyrate (GABA, see p. 356). The transmitter dopamine is also a precursor for the catecholamines epineph- rine and norepinephrine (see p. 352). The bio- genic amine serotonin, a substance that has many effects, is synthesized from tryptophan via the intermediate 5-hydroxytryptophan. Monamines are inactivated into aldehydes by amine oxidase (monoamine oxidase, “MAO”) with deamination and simultaneous oxidation. MAO inhibitors therefore play an important role in pharmacological interven- tions in neurotransmitter metabolism. 62 Biomolecules HN N COO C CH2 CH2 CH2 NH3 H3N H COO C CH2 CH2 CH2 N H3N H C NH2 O H COO C CH2 H3N H OH OH COO C CH2 H3N H Se H NH3COOCONH2OH SH OH OOC H3N Neurotrans- mitter (GABA) A. Rare amino acids D: Pyroglutamyl- Acetyl- Formyl- Myristoyl- B: Oligo- saccharide (O-glyco- sylation) B: Oligo- saccharide (N-glyco- sylation) D: Phospho- Methyl- γ-Carboxy- (Glu) D: Acetyl- Methyl- γ-Hydroxy- B: Pyridoxal- Liponat Biotin Retinal Ubiquitin Ser, Thr Asn, Gln Asp, Glu Lys Tyr Phe His Cys Pro Ornithine Citrulline L-Dopa Seleno- cysteine D: derivative B: bonds with Amine Function FunctionAmineAmino acid Serine Ethanol- amine Glutamate Cysteine Cysteamine Component of coenzyme A Threonine Amino- propanol Component of vitamin B12 Aspartate β-Alanine Component of coenzyme A γ-Amino- butyrate Histidine Mediator, neuro- transmitter Dopa Dopamine Neurotransmitter 5-Hydroxy- tryptophan Serotonin Mediator, neuro- transmitter Amino acid D: Disulfide Prenyl- B: Heme Flavin D: Phospho- Methyl- B: Flavin D: 4-Hydroxy- (Tyrosine) D: Phospho- Iodo- Sulfato- Adenyl- D: Amido- (CONH2) D: 3-Hydroxy- 4-Hydroxy- B. Post-translational protein modification C. Biogenic amines Glutamate Histamine 63Amino Acids Peptide bonds A. Peptide bond The amino acid components of peptides and proteins are linked together by amide bonds (see p. 60) between α-carboxyl and α-amino groups. This type of bonding is therefore also known as peptide bonding. In the dipeptide shown here, the serine residue has a free ammonium group, while the carboxylate group in alanine is free. Since the amino acid with the free NH3 + group is named first, the peptide is known as seryl alanine, or in abbre- viated form Ser-Ala or SA. B. Resonance Like all acid–amide bonds, the peptide bond is stabilized by resonance (see p. 4). In the con- ventional notation (top right) it is represented as a combination of a C=O double bond with a C–N single bond. However, a C=N double bond with charges at O and N could also be written (middle). Both of these are only extreme cases of electron distribution, known as resonance structures. In reality, the π electrons are delocalized throughout all the atoms (bot- tom). As a mesomeric system, the peptide bond is planar. Rotation around the C–N bond would only be possible at the expense of large amounts of energy, and the bond is therefore not freely rotatable. Rotations are only possible around the single bonds marked with arrows. The state of these is expressed using the angles φ and ψ (see D). The plane in which the atoms of the peptide bond lie is highlighted in light blue here and on the fol- lowing pages. C. Peptide nomenclature Peptide chains have a direction and therefore two different ends. The amino terminus (N terminus) of a peptide has a free ammonium group, while the carboxy terminus (C termi- nus) is formed by the carboxylate group of the last amino acid. In peptides and proteins, the amino acid components are usually linked in linear fashion. To express the sequence of a peptide, it is therefore suf cient to combine the three-letter or single-letter abbreviations for the amino acid residues (see p. 60). This sequence always starts at the N terminus. For example, the peptide hormone angiotensin II (see p. 330) has the sequence Asp-Arg-Val- Tyr-Ile-His-Pro-Phe, or DRVYIHPF. D. Conformational space of the peptide chain With the exception of the terminal residues, every amino acid in a peptide is involved in two peptide bonds (one with the preceding residue and one with the following one). Due to the restricted rotation around the C–N bond, rotations are only possible around the N–Cα and Cα–C bonds (2). As mentioned above, these rotations are described by the dihedral angles φ (phi) and ψ (psi). The angle describes rotation around the N–Cα bond; ψ describes rotation around Cα–C—i. e., the po- sition of the subsequent bond. For steric reasons, only specific combina- tions of the dihedral angles are possible. These relationships can be illustrated clearly by a so-called φ/ψ diagram (1). Most combi- nations of φ and ψ are sterically “forbidden” (red areas). For example, the combination φ = 0° and ψ = 180° (4) would place the two carbonyl oxygen atoms less than 115 pm apart—i. e., at a distance much smaller than the sum of their van der Waals radii (see p. 6). Similarly, in the case of φ = 180° and ψ = 0° (5), the two NH hydrogen atoms would collide. The combinations located within the green areas are the only ones that are sterically feasible (e. g., 2 and 3). The important secon- dary structures that are discussed in the fol- lowing pages are also located in these areas. The conformations located in the yellow areas are energetically less favorable, but still pos- sible. The φ/ψ diagram (also known as a Rama- chandran plot) was developed from modeling studies of small peptides. However, the con- formations of most of the amino acids in pro- teins are also located in the permitted areas. The corresponding data for the small protein, insulin (see p. 76), are represented by black dots in 1. 66 Biomolecules 180 120 60 ψ 0 -60 -120 -180 -120 -60 0 60 120 180 ϕ A-9 B-20 B-8 αr βp βa C B-23 αl 2. 3. 4. 5. ϕ ψ 2 R 1 3 R 3 1 ϕ ψ2 R ϕ ψ 1 3 2 ϕ ψ R 2 1 3 1. C N H O C N H O C N H O ϕ ψ H3N C C N C C N C C N C C N C COO R1 O H R2 O H R3 O H R4 O H Rn HHH H H C N H O C N O H C N O H A. Peptide bonds C. Peptide nomenclature Residue 1 Residue 2 Residue 3 Residue 4 Residue Amino terminus (N terminus) Carboxy- terminus (C terminus) B. Resonance Resonance structures Mesomeric structure Seryl alanine (Ser-Ala, H3N-Ser-Ala-COO , SA) D. Conformation space of the peptide chain ϕ (Phi): Rotation about j (Phi): N – Cαψ (Psi): Rotation about y (Psi): Cα – C Allowed Forbidden ϕ = 0° ψ = 180° d = 115 pm ϕ = -139° ψ = -135° ϕ = 180° ψ = 0° d = 155 pm ϕ = -57° ψ = -47° Collagen helixC Pleated sheet (antiparallel) Pleated sheet (parallel) αr αl α Helix (right-handed) α Helix (left-handed) βa βp 67Peptides and Proteins Secondary structures In proteins, specific combinations of the dihe- dral angles φ and ψ (see p. 66) are much more common than others. When several succes- sive residues adopt one of these conforma- tions, defined secondary structures arise, which are stabilized by hydrogen bonds ei- ther within the peptide chain or between neighboring chains. When a large part of a protein takes on a defined secondary struc- ture, the protein often forms mechanically stable filaments or fibers. Structural proteins of this type (see p. 70) usually have character- istic amino acid compositions. The most important secondary structural elements of proteins are discussed here first. The illustrations only show the course of the peptide chain; the side chains are omitted. To make the course of the chains clearer, the levels of the peptide bonds are shown as blue planes. The dihedral angles of the struc- tures shown here are also marked in diagram D1 on p. 67. A. -Helix The right-handed α-helix (αR) is one of the most common secondary structures. In this conformation, the peptide chain is wound like a screw. Each turn of the screw (the screw axis in shown in orange) covers approxi- mately 3.6 amino acid residues. The pitch of the screw (i. e., the smallest distance between two equivalent points) is 0.54 nm. α-Helices are stabilized by almost linear hydrogen bonds between the NH and CO groups of residues, which are four positions apart from each an- other in the sequence (indicated by red dots; see p. 6). In longer helices, most amino acid residues thus enter into two H bonds. Apolar or amphipathic α-helices with five to seven turns often serve to anchor proteins in bio- logical membranes (transmembrane helices; see p. 214). The mirror image of the αR helix, the left- handed -helix (αL), is rarely found in nature, although it would be energetically “permissi- ble.” B. Collagen helix Another type of helix occurs in the collagens, which are important constituents of the con- nectivetissue matrix (see pp. 70, 344). The collagen helix is left-handed, and with a pitch of 0.96 nm and 3.3 residues per turn, it is steeper than the α-helix. In contrast to the α-helix, H bonds are not possible within the collagen helix. However, the conformation is stabilized by the association of three helices to form a righthanded collagen triple helix (see p. 70). C. Pleated-sheet structures Two additional, almost stretched, conforma- tions of the peptide chain are known as pleated sheets, as the peptide planes are ar- ranged like a regularly folded sheet of paper. Again, H bonds can only form between neigh- boring chains (“strands”) in pleated sheets. When the two strands run in opposite direc- tions (1), the structure is referred to as an antiparallel pleated sheet (βa). When they run in the same direction (2), it is a parallel pleated sheet (βp). In both cases, the α-C atoms occupy the highest and lowest points in the structure, and the side chains point alternately straight up or straight down (see p. 71 C). The βa structure, with its almost lin- ear H bonds, is energetically more favorable. In extended pleated sheets, the individual strands of the sheet are usually not parallel, but twisted relative to one another (see p. 74). D. Turns Turns are often found at sites where the peptide chain changes direction. These are sections in which four amino acid residues are arranged in such a way that the course of the chain reverses by about 180° into the opposite direction. The two turns shown (types I and II) are particularly frequent. Both are stabilized by hydrogen bonds be- tween residues 1 and 4. β Turns are often located between the individual strands of antiparallel pleated sheets, or between strands of pleated sheets and α helices. 68 Biomolecules Ala Gly Ala Gly Ala Ala Gly Gly GlyGly SerAla Gly Gly Gly Gly Gly Gly Arg Gln Pro Pro Ala X Hyp Arg Hyp Gln Arg Y Gly 3 nm 10 nm A. α-Keratin C. Silk fibroin Left-handed superhelix Right-handed α helix Intermediary filament Protofilament 1. Spatial illustration B. Collagen 1. Triple helix (section) 2. Front view 0. 35 n m 0. 57 n m 3. Triple helix (view from above) 2. Typical sequence 71Peptides and Proteins Globular proteins Soluble proteins have a more complex struc- ture than the fibrous, completely insoluble structural proteins. The shape of soluble pro- teins is more or less spherical (globular). In their biologically active form, globular proteins have a defined spatial structure (the native conformation). If this structure is destroyed (denaturation; see p. 74), not only does the biological effect disappear, but the protein also usually precipitates in insoluble form. This happens, for example, when eggs are boiled; the proteins dissolved in the egg white are denatured by the heat and produce the solid egg white. To illustrate protein conformations in a clear (but extremely simplified) way, Richard- son diagrams are often used. In these diagrams, α-helices are symbolized by red cylinders or spirals and strands of pleated sheets by green arrows. Less structured areas of the chain, including the β-turns, are shown as sections of gray tubing. A. Conformation-stabilizing interactions The native conformation of proteins is stabi- lized by a number of different interactions. Among these, only the disulfide bonds (B) represent covalent bonds. Hydrogen bonds, which can form inside secondary structures, as well as between more distant residues, are involved in all proteins (see p. 6). Many pro- teins are also stabilized by complex formation with metal ions (see pp. 76, 342, and 378, for example). The hydrophobic effect is particu- larly important for protein stability. In glob- ular proteins, most hydrophobic amino acid residues are arranged in the interior of the structure in the native conformation, while the polar amino acids are mainly found on the surface (see pp. 28, 76). B. Disulfide bonds Disulfide bonds arise when the SH groups of two cysteine residues are covalently linked as a dithiol by oxidation. Bonds of this type are only found (with a few exceptions) in extra- cellular proteins, because in the interior of the cell glutathione (see p. 284) and other reduc- ing compounds are present in such high con- centrations that disulfides would be reduc- tively cleaved again. The small plant protein crambin (46 amino acids) contains three di- sulfide bonds and is therefore very stable. The high degree of stability of insulin (see p. 76) has a similar reason. C. Protein dynamics The conformations of globular proteins are not rigid, but can change dramatically on binding of ligands or in contact with other proteins. For example, the enzyme adenylate kinase (see p. 336) has a mobile domain (do- main = independently folded partial struc- ture), which folds shut after binding of the substrate (yellow). The larger domain (bot- tom) also markedly alters its conformation. There are large numbers of allosteric proteins of this type. This group includes, for example, hemoglobin (see p. 280), calmodulin (see p. 386), and many allosteric enzymes such as aspartate carbamoyltransferase (see p.116). D. Folding patterns The globular proteins show a high degree of variability in folding of their peptide chains. Only a few examples are shown here. Purely helically folded proteins such as myoglobin (1; see p. 74, heme yellow) are rare. In general, pleated sheet and helical elements exist alongside each other. In the hormone-binding domain of the estrogen receptor (2; see p. 378), a small, two-stranded pleated sheet functions as a “cover” for the hormone binding site (estradiol yellow). In flavodoxin, a small flavo- protein with a redox function (3; FMN yel- low), a fan-shaped, pleated sheet made up of five parallel strands forms the core of the molecule. The conformation of the β subunit of the G-protein transducin (4; see pp. 224, 358) is very unusual. Seven pleated sheets form a large, symmetrical “β propeller.” The N-terminal section of the protein contains one long and one short helix. 72 Biomolecules S NH CO SS C O N H CH2HC CH2 CH A. Conformation-stabilizing interactions C. Protein dynamics D. Folding patterns 4. Transducin (β subunit) B. Disulfide bonds 1. Myoglobin 2. Estrogen receptor (domain) 3. Flavodoxin Polar surface Apolar core Disulfide bond Metal complex Hydrogen bond Mobile domain Adenylate kinase Substrate 73Peptides and Proteins Molecular models: insulin The opposite page presents models of insulin, a small protein. The biosynthesis and function of this important hormone are discussed else- where in this book (pp.160, 388). Monomeric insulin consists of 51 amino acids, and with a molecular mass of 5.5 kDa it is only half the size of the smallest enzymes. Nevertheless, it has the typical properties of a globular pro- tein. Large quantities of pure insulin are re- quired for the treatment of diabetes mellitus (see p.160). The annual requirement for insu- lin is over 500 kg in a country the size of Germany. Formerly, the hormone had to be obtained from the pancreas of slaughtered animals in a complicated and expensive procedure. Human insulin, which is produced by overexpression in genetically engineered bacteria, is now mainly used (see p. 262). A. Structure of insulin There are various different structural levels in proteins, and these can be briefly discussed again here using the example of insulin. The primary structure of a protein is its amino acid sequence. During the biosynthesis of insulin in the pancreas, a continuous pep- tide chain with 84 residues is first synthesi- zed—proinsulin (see p.160). After folding of the molecule, the three disulfide bonds are first formed, and residues 31 to 63 are then proteolytically cleaved releasing the so-called C peptide. The molecule that is left over (1) now consists of two peptide chains, the A chain (21 residues, shown in yellow) and the B chain (30 residues, orange). One of the di- sulfide bonds is located inside the A chain, and the two others link the two chains to- gether. Secondary structures are regions of the peptide chain with a defined conformation (see p. 68) that are stabilized by H-bonds. In insulin (2), the α-helical areas are predomi- nant, making up 57% of the molecule; 6% consists of β-pleated-sheet structures, and 10% of β-turns, while the remainder (27%) cannot be assigned to any of the secondary structures. The three-dimensional conformation of a protein, made up of secondary structural ele- ments and unordered sections, is referred to as the tertiary structure. In insulin, it is com- pact and wedge-shaped (B). The tip of the wedge is formed by the B chain, which changes its direction at this point. Quaternary structure. Due to non-covalent interactions, many proteins assemble to form symmetrical complexes (oligomers). The indi- vidual components of oligomeric proteins (usually 2–12) are termed subunits or mono- mers. Insulin also forms quaternary struc- tures. In the blood, it is partly present as a dimer. In addition, there are also hexamers stabilized by Zn2+ ions (light blue) (3), which represent the form in which insulin is stored in the pancreas (see p.160). B. Insulin (monomer) The van der Waals model of monomeric in- sulin (1) once again shows the wedge-shaped tertiary structure formed by the two chains together. In the second model (3, bottom), the side chains of polar amino acids are shown in blue, while apolar residues are yellow or pink. This model emphasizes the importance of the “hydrophobic effect” for protein folding (see p. 74). In insulin as well, most hydrophobic side chains are located on the inside of the molecule, while the hydrophilic residues are located on the surface. Apparently in contra- diction to this rule, several apolar side chains (pink) are found on the surface. However, all of these residues are involved in hydrophobic interactions that stabilize the dimeric and hexameric forms of insulin. In the third model (2, right), the colored residues are those that are located on the surface and occur invariably (red) or almost invariably (orange) in all known insulins. It is assumed that amino acid residues that are not replaced by other residues during the course of evolution are essential for the protein’s function. In the case of insulin, almost all of these residues are located on one side of the molecule. They are probably involved in the binding of the hormone to its receptor (see p. 224). 76 Biomolecules 2324252627282930 R R A K P T Y F F G R 22 1 2 3 4 5 6 12 13 14 15 16 17 18 19 20 CG I V E Q S L Y Q L E N 21 Y C N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 F V N Q H L C G S H L V E A L Y L V C G E T S I 7 C 11 C 8 9 10 K R Q E NH3 NH3 OOC OOC NH3 OOC OOC NH3 1. 2. 3. A chain C peptide 2. Secondary and tertiary structure B chain A-chain B-chain Polar side chain Apolar side chain Invariant residue Involved in subunit interactions A. Structure of insulin 1. Primary structure B chain A chain C Peptide Disulfide bonds 3. Quaternary structure B. Insulin (monomer) 77Peptides and Proteins Isolation and analysis of proteins Purified proteins are nowadays required for a wide variety of applications in research, med- icine, and biotechnology. Since the globular proteins in particular are very unstable (see p. 72), purification is carried out at low tem- peratures (0–5 °C) and particularly gentle separation processes are used. A few of the methods of purifying and characterizing pro- teins are discussed on this page. A. Salt precipitation The solubility of proteins is strongly depen- dent on the salt concentration (ionic strength) of the medium. Proteins are usually poorly soluble in pure water. Their solubility in- creases as the ionic strength increases, be- cause more and more of the well-hydrated anorganic ions (blue circles) are bound to the protein’s surface, preventing aggregation of the molecules (salting in). At very high ionic strengths, the salt withdraws the hy- drate water from the proteins and thus leads to aggregation and precipitation of the mole- cules (salting out). For this reason, adding salts such as ammonium sulfate (NH4)2SO4 makes it possible to separate proteins from a mixture according to their degree of solubility (fractionation). B. Dialysis Dialysis is used to remove lower-molecular components from protein solutions, or to ex- change the medium. Dialysis is based on the fact that due to their size, protein molecules are unable to pass through the pores of a semipermeable membrane, while lower-mo- lecular substances distribute themselves evenly between the inner and outer spaces over time. After repeated exchanging of the external solution, the conditions inside the dialysis tube (salt concentration, pH, etc.) will be the same as in the surrounding solu- tion. C. Gel filtration Gel permeation chromatography (“gel filtra- tion”) separates proteins according to their size and shape. This is done using a chroma- tography column, which is filled with spherical gel particles (diameter 10–500 µm) of polymeric material (shown schematically in 1a). The insides of the particles are tra- versed by channels that have defined diame- ters. A protein mixture is then introduced at the upper end of the column (1b) and elution is carried out by passing a buffer solution through the column. Large protein molecules (red) are unable to penetrate the particles, and therefore pass through the column quickly. Medium-sized (green) and small par- ticles (blue) are delayed for longer or shorter periods (1c). The proteins can be collected separately from the ef uent (eluate) (2). Their elution volume Ve depends mainly on their molecular mass (3). D. SDS gel electrophoresis The most commonly used procedure for checking the purity of proteins is sodium do- decyl sulfate polyacrylamide gel electropho- resis (SDS-PAGE). In electrophoresis, mole- cules move in an electrical field (see p. 276). Normally, the speed of their movement de- pends on three factors—their size, their shape, and their electrical charge. In SDS-PAGE, the protein mixture is treated in such a way that only the molecules’ mass affects their movement. This is achieved by adding sodium dodecyl sulfate (C12H25- OSO3Na), the sulfuric acid ester of lauryl alco- hol (dodecyl alcohol). SDS is a detergent with strongly amphipathic properties (see p. 28). It separates oligomeric proteins into their sub- units and denatures them. SDS molecules bind to the unfolded peptide chains (ca. 0.4 g SDS / g protein) and give them a strongly negative charge. To achieve complete denatu- ration, thiols are also added in order to cleave the disulfide bonds (1). Following electrophoresis, which is carried out in a vertically arranged gel of polymeric acrylamide (2), the separated proteins are made visible by staining. In example (3), the following were separated: a) a cell extract with hundreds of different proteins, b) a pro- tein purified from this, and c) a mixture of proteins with known masses. 78 Biomolecules 1 2 3 4 56 7 9 1 2 3 4 5 6 8 RNA F A F A U G T P T P A 5' 1' β A 5' 1' β N HC N CH CH H C HN C N H CH CH C O O HN C N H CH C C O O CH3 N C N H CH CH C O NH2 N HC N C C H C N H CH N N HC N C C C N H CH N NH2 N CH N C C C N HC N NH2 O CH2HO H OH OH H H H N O CH2O H OH H H H H PO O O CH C C HN C O CH3 O N C N C C C N C N N O CH2O H OH OH H H H H H H H PO O O PO O O H2C CH OH C C OHH CH2 OHH N C C N C C C C C C N C N CH3C H3C H H O H O NH C N C C C N HC N O O CH2O H O OH H H H NH2PO O O N O CH2O H OH H H H PO O O C NH C HC HC O O To the 5' end A. Nucleic acid bases Pyrimidine Pyrimidine bases Purine bases Purine Uracil (Ura) Thymine (Thy) Cytosine (Cyt) Adenine (Ade) Guanine (Gua) B. Nucleosides, nucleotides 1. Adenosine (Ado) 2. 2'-Deoxythymidine 5'-monophosphate (dtMP) C. Oligonucleotides, polynucleotides 1. Flavin adenine dinucleotide (FAD) 2. RNA (section) Flavin To the 3' end Ribose Ribitol Phosphoric acid diester bond Phosphoric acid– anhydride bond HN C N C C C N H CH N O H2N 81Nucleotides and Nucleic Acids RNA Ribonucleic acids (RNAs) are polymers con- sisting of nucleoside phosphate components that are linked by phosphoric acid diester bonds (see p. 80). The bases the contain are mainly uracil, cytosine, adenine, and guanine, but many unusual and modified bases are also found in RNAs (B). A. Ribonucleic acids (RNAs) RNAs are involved in all the individual steps of gene expression and protein biosynthesis (see pp. 242–253). The properties of the most im- portant forms of RNA are summarized in the table. The schematic diagram also gives an idea of the secondary structure of these mol- ecules. In contrast to DNA, RNAs do not form ex- tended double helices. In RNAs, the base pairs (see p. 84) usually only extend over a few residues. For this reason, substructures often arise that have a finger shape or clover-leaf shape in two-dimensional representations. In these, the paired stem regions are linked by loops. Large RNAs such as ribosomal 16S- rRNA (center) contain numerous “stem and loop” regions of this type. These sections are again folded three-dimensionally—i. e., like proteins, RNAs have a tertiary structure (see p. 86). However, tertiary structures are only known of small RNAs, mainly tRNAs. The dia- grams in Fig. B and on p. 86 show that the “clover-leaf” structure is not recognizable in a three-dimensional representation. Cellular RNAs vary widely in their size, structure, and lifespan. The great majority of them are ribosomal RNA (rRNA), which in several forms is a structural and functional component of ribosomes (see p. 250). Riboso- mal RNA is produced from DNA by transcrip- tion in the nucleolus, and it is processed there and assembled with proteins to form ribo- some subunits (see pp. 208, 242). The bacte- rial 16S-rRNA shown in Fig. A, with 1542 nu- cleotides (nt), is a component of the small ribosomae subunit, while the much smaller 5S-rRNA (118 nt) is located in the large sub- unit. Messenger RNAs (mRNAs) transfer genetic information from the cell nucleus to the cyto- plasm. The primary transcripts are substan- tially modified while still in the nucleus (mRNA maturation; see p. 246). Since mRNAs have to be read codon by codon in the ribo- some, they must not form a stable tertiary structure. This is ensured in part by the at- tachment of RNA-binding proteins, which pre- vent base pairing. Due to the varying amounts of information that they carry, the lengths of mRNAs also vary widely. Their lifespan is usu- ally short, as they are quickly broken down after translation. Small nuclear RNAs (snRNAs) are involved in the splicing of mRNA precursors (see p. 246). They associate with numerous pro- teins to form “spliceosomes.” B. Transfer RNA (tRNAPhe) The transfer RNAs (tRNAs) function during translation (see p. 250) as links between the nucleic acids and proteins. They are small RNA molecules consisting of 70–90 nucleoti- des, which “recognize” specific mRNA codons with their anticodons through base pairing. At the same time, at their 3 end (sequence .. CCA-3) they carry the amino acid that is assigned to the relevant mRNA codon accord- ing to the genetic code (see p. 248). The base sequence and the tertiary struc- ture of the yeast tRNA specific for phenylala- nine (tRNAPhe) is typical of all tRNAs. The molecule (see also p. 86) contains a high pro- portion of unusual and modified components (shaded in dark green in Fig. 1). These include pseudouridine (Ψ), dihydrouridine (D), thymi- dine (T), which otherwise only occurs in DNA, and many methylated nucleotides such as 7- methylguanidine (m7G) and—in the anti- codon—2-O-methylguanidine (m2G). Numer- ous base pairs, sometimes deviating from the usual pattern, stabilize the molecule’s confor- mation (2). 82 Biomolecules 3' 5' 3'5' 3' 5' 3' 5' 3' 5' * 3' 5' * * * * * * * PheA C G U C A C U A A G A AC C G UUCC U G GAG C G U A Y Y G A A U C C A G A C G G A G C AG G G D D G A C U C G A U U U A G G C G G C T C U A G CY Phe 3' 5' NH C N C C C N HC N O O CH2O H O OH H H H NH CH3 1' 3' O N O CH2O H OH H H H C NH C H2C H2C O O O C O CH2O H OH H H H C NH C HN HC O O5' NH C N C C C N HC N O O CH2O H O OCH3 H H H NH2 A. Ribonucleic acids (RNAs) B. Transfer RNA (tRNAPhe) tRNA 16S-RNA 5S-rRNA mRNA U1-snRNA 1. Structure Dihydrouridine (D) Pseudouridine (Ψ) 2. Conformation D loop TΨ loop Variable loop mRNA Codon Species per cell Type Length (b) Proportion Lifespan Function Variable loop Anticodon TΨ loop D loop 7-methylguanidine (m7G)2'-O-methylguanidine (m2G) Normal base pairing Unusual base pairing >50 4 > 1000 ~ 10 tRNA rRNA mRNA snRNA 74 - 95 120 - 5000 400 - 6000 100 - 300 10-20% 80% 5% < 1% Long Long Short Long Translation Translation Translation Splicing * Methylated base Anticodon 83Nucleic Acids Molecular models: DNA and RNA The illustration opposite shows selected nuc- leic acid molecules. Fig. A shows various con- formations of DNA, and Fig. B shows the spa- tial structures of two small RNA molecules. In both, the van der Waals models (see p. 6) are accompanied by ribbon diagrams that make the course of the chains clear. In all of the models, the polynucleotide “backbone” of the molecule is shown in a darker color, while the bases are lighter. A. DNA: conformation Investigations of synthetic DNA molecules have shown that DNA can adopt several dif- ferent conformations. All of the DNA seg- ments shown consist of 21 base pairs (bp) and have the same sequence. By far the most common form is B-DNA (2). As discussed on p. 84, this consists of two antiparallel polydeoxynucleotide strands in- tertwined with one another to form a right- handed double helix. The “backbone” of these strands is formed by deoxyribose and phos- phate residues linked by phosphoric acid di- ester bonds. In the B conformation, the aromatic rings of the nucleobases are stacked at a distance of 0.34 nm almost at right angles to the axis of the helix. Each base is rotated relative to the preceding one by an angle of 35°. A complete turn of the double helix (360°) therefore con- tains around 10 base pairs (abbreviation: bp), i. e., the pitch of the helix is 3.4 nm. Between the backbones of the two individual strands there are two grooves with different widths. The major groove is visible at the top and bottom, while the narrower minor groove is seen in the middle. DNA-binding proteins and transcription factors (see pp.118, 244) usually enter into interactions in the area of the major groove, with its more easily accessible bases. In certain conditions, DNA can adopt the A conformation (1). In this arrangement, the double helix is still right-handed, but the bases are no longer arranged at right angles to the axis of the helix, as in the B form. As can be seen, the A conformation is more compact than the other two conformations. The minor groove almost completely disappears, and the major groove is narrower than in the B form. A-DNA arises when B-DNA is dehydrated. It probably does not occur in the cell. In the Z-conformation (3), which can occur within GC-rich regions of B-DNA, the organ- ization of the nucleotides is completely differ- ent. In this case, the helix is left-handed, and the backbone adopts a characteristic zig-zag conformation (hence “Z-DNA”). The Z double helix has a smaller pitch than B-DNA. DNA segments in the Z conformation probably have physiological significance, but details are not yet known. B. RNA RNA molecules are unable to form extended double helices, and are therefore less highly ordered than DNA molecules. Nevertheless, they have defined secondary and tertiary structures, and a large proportion of the nu- cleotide components enter into base pairings with other nucleotides. The examples shown here are 5S-rRNA (see p. 242), which occurs as a structural component in ribosomes, and a tRNA molecule from yeast (see p. 82) that is specific for phenylalanine. Both molecules are folded in such a way that the 3 end and the 5 end are close to- gether. As in DNA, most of the bases are lo- cated in the inside of the structures, while the much more polar “backbone” is turned out- wards. An exception to this is seen in the three bases of the anticodon of the tRNA (pink), which have to interact with mRNA and therefore lie on the surface of the mole- cule. The bases of the conserved CCA triplet at the 3 end (red) also jut outward. During amino acid activation (see p. 248), they are recognized and bound by the ligases. 86 Biomolecules 5'3' 5'3' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' 5'3' A. DNA: conformation B. RNA 1. A - DNA 2. B - DNA 3. Z - DNA Backbone Bases 1. 5S-rRNA 2. Phe-tRNAPhe (118 nucleotides) (77 nucleotides) 87Nucleic Acids Enzymes: basics Enzymes are biological catalysts—i. e., sub- stances of biological origin that accelerate chemical reactions (see p. 24). The orderly course of metabolic processes is only possible because each cell is equipped with its own genetically determined set of enzymes. It is only this that allows coordinated sequences of reactions (metabolic pathways; see p.112). Enzymes are also involved in many regulatory mechanisms that allow the metabolism to adapt to changing conditions (see p.114). Al- most all enzymes are proteins. However, there are also catalytically active ribonucleic acids, the “ribozymes” (see pp. 246, 252). A. Enzymatic activity The catalytic action of an enzyme, its activity, is measured by determining the increase in the reaction rate under precisely defined con- ditions—i. e., the difference between the turn- over (violet) of the catalyzed reaction (or- ange) and uncatalyzed reaction (yellow) in a specific time interval. Normally, reaction rates are expressed as the change in concentration per unit of time (mol 1–1 s–1; see p. 22). Since the catalytic activity of an enzyme is independent of the volume, the unit used for enzymes is usually turnover per unit time, expressed in katal (kat, mol s–1). However, the international unit U is still more com- monly used (µmol turnover min–1; 1 U = 16.7 nkat). B. Reaction and substrate specificity The action of enzymes is usually very specific. This applies not only to the type of reaction being catalyzed (reaction specificity), but also to the nature of the reactants (“substrates”) that are involved (substrate specificity; see p. 94). In Fig. B, this is illustrated schemati- cally using a bond-breaking enzyme as an example. Highly specific enzymes (type A, top) catalyze the cleavage of only one type of bond, and only when the structure of the substrate is the correct one. Other enzymes (type B, middle) have narrow reaction specif- icity, but broad substrate specificity. Type C enzymes (with low reaction specificity and low substrate specificity, bottom) are very rare. C. Enzyme classes More than 2000 different enzymes are cur- rently known. A system of classification has been developed that takes into account both their reaction specificity and their substrate specificity. Each enzyme is entered in the En- zyme Catalogue with a four-digit Enzyme Commission number (EC number). The first digit indicates membership of one of the six major classes. The next two indicate sub- classes and subsubclasses. The last digit indi- cates where the enzyme belongs in the sub- subclass. For example, lactate dehydrogenase (see pp. 98–101) has the EC number 1.1.1.27 (class 1, oxidoreductases; subclass 1.1, CH–OH group as electron donor; sub-subclass 1.1.1, NAD(P)+ as electron acceptor). Enzymes with similar reaction specificities are grouped into each of the six major classes: The oxidoreductases (class 1) catalyze the transfer of reducing equivalents from one re- dox system to another. The transferases (class 2) catalyze the transfer of other groups from one molecule to another. Oxidoreductases and transferases generally require coenzymes (see pp.104ff.). The hydrolases (class 3) are also involved in group transfer, but the acceptor is always a water molecule. Lyases (class 4, often also referred to as “synthases”) catalyze reactions involving ei- ther the cleavage or formation of chemical bonds, with double bonds either arising or disappearing. The isomerases (class 5) move groups within a molecule, without changing the gross composition of the substrate. The ligation reactions catalyzed by ligases (“synthetases,” class 6) are energy-dependent and are therefore always coupled to the hy- drolysis of nucleoside triphosphates. In addition to the enzyme name, we also usually give its EC number. The annotated enzyme list (pp. 420ff.) includes all of the en- zymes mentioned in this book, classified ac- cording to the Enzyme Catalog system. 88 Metabolism