The Substances of Life

3 THE SUBSTANCES OF LIFE A useful simplification of biological organisms sometimes made by environmental engineers and scientists is to view them as catalysts for chemical reactions, such as the oxidation of ammonia or ferrous iron, or production of methane and carbon dioxide from acetic acid. Such a view hides the detailed mechanisms, including the sequence of chemical intermediates and the specific chemical nature of the catalyst. Examining these details will help us to understand more complex chemical interactions between organisms and their environment, such as biodegradation of toxic organic chemicals or the effect of chemicals on the health of organisms and ecosystems. The details of biochemistry begin with knowledge of the four most important types of chemical substances comprising living things: carbohydrates, lipids, proteins, and nucleic acids. Later, in the chapters on toxicology, we consider the biochemical reactions involving xenobiotic compounds (those that are, literally, ‘‘foreign to life,’’ that is, substances not normally found in living things). 3.1 BASIC ORGANIC CHEMICAL STRUCTURE A fairly simple summary of organic chemistry is sufficient for understanding much of the structure and reactions of biochemicals. There are only two types of chemical bonds that need to be considered at this point: ionic bonds and covalent bonds. The great majority of biochemicals are composed of only six elements connected by those bonds: carbon, hydrogen, nitrogen, oxygen, sulfur, and phosphorus. In addition, the attractions of dipole forces such as hydrogen bonds and van der Waals forces act between molecules or between different parts of the same molecule. The structure of most organic chemicals can be described by combining the six elements with ionic and covalent chemical bonds. 35 36 THE SUBSTANCES OF LIFE Physicochemical interactions are attractions or repulsions between molecules, or between different parts of the same molecule, which do not result in formation or breaking of ionic or covalent bonds. They are much weaker than chemical bonds and are much more sensitive to changes in temperature. These forces change the shape of molecules, causing them to bend, fold, or form liquids or crystals. This affects physical properties such as solubility or boiling point as well as the ability of molecules to enter into chemical reactions. Much of this physicochemical behavior can be related to a few types of atomic groups on the molecule that are called functional groups. The main importance of this section is to arrive at an understanding of what controls the shape of biochemical compounds. Biochemicals are often large, polymeric molecules, whose function is intimately related to their shape. Thus, their behavior is not determined completely by their chemical formula, but also depends on how they are folded or coiled or by how they are arranged relative to neighboring molecules. 3.2 CHEMICAL BONDING Atoms in a molecule are held together by covalent or ionic bonds. These bonds involve a sharing of electrons between pairs of atoms. Bonds can be classified in terms of how equally the electrons are shared or by the amount of energy required to break the bond. The electrons in covalent bonds are equally or almost equally shared, whereas those in ionic bonds are associated almost completely with one of the atoms in the bonded pair. Electrons in bonds between identical atoms tend to be shared equally between the two. But if the two atoms are different, the electron cloud making up the bond may be displaced toward one of them. As a result, the center of charge of the electrons is different from the center of charge of the nuclei of the atoms, and the bond will produce an electrostatic field in its vicinity. A bond that produces an electrostatic field is said to be polar. Polarity produces attractions between molecules. For example, Figure 3.1 shows an ethylene molecule. The bond between the two carbons will be nonpolar. A molecule of ethanol is also shown. The carbon and oxygen atoms CH2 CH3 CH2 H2C Ethylene OH Ethanol COOH HC CH2 Proprionic acid OH Lactic acid HC O NH2 HC OH CH2 H2C OH COOH Glyceraldehyde Glycine Figure 3.1 Oxalic acid CH3 COOH COOH COOH Acetic acid CH3 CH3 COOH CH3 H2C OH O HC OH COOH H2C OH Pyruvic acid Glycerol C NH2 NH2 HC CH3 C O COOH NH2 Alanine Urea Structures of several small, biologically important organic molecules. CHEMICAL BONDING 37 are very different in their tendency to attract electrons; thus, this molecule is polar. The tendency of atoms to attract electrons is called its electronegativity. Oxygen is more electronegative than carbon; therefore, the ethanol molecule will be polarized with a positive charge toward the carbon and negative charge toward the oxygen. Sometimes oxygen can attract electrons away from a neighboring hydrogen so completely that it leaves the hydrogen fairly free to dissociate from the molecule as a proton, producing an acid. This is seen most commonly in organic chemicals in the case of the carboxylic acid group: O O C C OH O− + H+ also written

COOH. The two oxygen atoms act together to strip the electron from the hydrogen. Fluorine is the most electronegative of all the elements. Figure 3.2 shows the electronegativity of all the elements that have been shown to be essential in at least some living things. The electronegativity of the other elements (except the noble elements) tends to decrease with distance from fluorine in the periodic table. Thus, chlorine is somewhat less electronegative than fluorine. The scale decreases more rapidly by moving to the left; thus, oxygen, nitrogen, and carbon represent a sequence of decreasing affinities for electrons. The leftmost column contains the alkali elements that are so low in electron affinity that they tend to give up their electrons entirely in bonds formed with the halogens on the right. These form the most polar of the bonds, the ionic bond. The symbol-and-stick diagram for atoms and bonds in Figure 3.1 shows features that can be used to recognize the covalent and ionic bonding arrangement for the vast majority of organic compounds. Atoms form bonds in order to give themselves enough electrons to fill an orbital shell, such as the noble elements have. In simpler terms, each element generally shares as many electrons (i.e., forms as many bonds) as corresponds to the number of columns that element is away from a noble element in the periodic table. For example, oxygen is two columns away from argon, so it forms two bonds and should be drawn as

O

or

O, the latter showing that two single bonds can be combined into a double bond. Carbon, nitrogen, and phosphorus can also form triple bonds, although the amount H 2.1 Li 1.0 Na 0.9 K 0.8 Be 1.5 Mg 1.2 Ca 1.0 Sc 1.3 Ti 1.5 V 1.6 Cr 1.6 Mo 1.8 W 1.7 Mn 1.5 Fe 1.8 Co 1.8 Ni 1.8 Cu 1.9 Zn 1.6 B 2.0 Al 1.5 Ga 1.6 C 2.5 Si 1.8 Ge 1.8 Sn 1.8 N 3.0 P 2.1 As 1.9 O 3.5 S 2.5 Se 2.4 F 4.0 Cl 3.0 Br 2.8 I 2.5 Figure 3.2 Electronegativity of biologically important elements. This shows the portion of the periodic table containing many of the elements that are important to life. The numbers indicate the electronegativity of the corresponding elements. (From Pauling, 1970.) 38 THE SUBSTANCES OF LIFE TABLE 3.1 Number of Bonds Commonly Formed by the Six Principal Elements of Biochemical Compounds Element Number of bonds C 4 N 3 O 2 S 2 P 5 H 1 of energy required makes these uncommon. Table 3.1 shows the number of covalent or ionic bonds formed by the six principal elements. There are several important exceptions to this simple scenario for an element that forms the number of bonds corresponding to its valence state (see Table 13.1). One of these exceptions is the metals. For example, iron forms two bonds in the ferrous (II) state and three in the ferric (III) state. Sulfur typically forms two bonds in organic compounds but can have other numbers in inorganics. Even carbon has exceptions to the rule, as in the case of carbon monoxide. Also, phosphorus is almost always bound in the phosphate group (PO3
4 ), which is usually considered as a unit. Ionic bonds are much weaker than covalent bonds. As a result, the atoms in ionic bonds can be separated by the relatively low-energy physicochemical forces. Although ionic and covalent bonds seem quite distinct, there is actually a continuum of bonds of varying polarity from ionic to covalent. 3.3 ACID–BASE REACTIONS A special type of ionic bond is when one of the ions is either Hþ or OH
, resulting in an acid or a base, respectively. Water serves as a source and sink for hydrogen ions that are generated or consumed by the dissociation of acids and bases. In fact, the very ability of other acids in solution to act as acids may depend on their being dissolved in water. Put another way, the presence of water shifts the acid dissociation equilibrium. The prototypical acid–base reaction is HA , Hþ þ A
ð3:1Þ where HA is the undissociated acid and A
is its conjugate base. The equilibrium constant is called the acid dissociation constant, Ka (square brackets denote molar concentration): Ka ¼ ½Hþ ½A
 ½HA ð3:2Þ Taking the logarithm of equation (3.2), substituting the relations pH ¼
log[Hþ] and pKa ¼
log½Ka , and rearranging, we obtain an expression for the fraction of undissociated acid: ½HA 1
¼ ½HA þ ½A  1 þ 10pH
pKa ð3:3Þ Thus, the relative proportion of HA and A
depends on the pH. At low pH most of the acid is undissociated. At high pH most is dissociated. The pKa is the pH at which the ACID–BASE REACTIONS 39 Fraction Undissociated 1.25 1.00 0.75 0.50 0.25 pKa 0.00 0.0 2.0 4.0 pH 6.0 8.0 Figure 3.3 Fraction of undissociated acid for acetic acid (pKa ¼ 4:7). Points shown are for pH 3.7, 4.7, and 5.7. equilibrium is positioned at the 50:50 point: half as HA and half as A
. Figure 3.3 shows a plot of the fraction ½HA=CT for acetic acid, where CT ¼ ½HA þ ½A
 is the total acid concentration. Table 3.2 shows pKa values for some important chemicals. Note that at 1 or 2 pH units below pKa , the acid is almost completely undissociated, whereas at 1 or 2 units above pKa , it is almost completely dissociated. Example 3.1 What fraction, f, of acetic acid is undissociated at pH 7.0? The pKa is 4.7; thus, the fraction undissociated is f ¼ 1 1 ¼ 0:50% ¼ 7:0
4:7 1 þ 10 1 þ 200 The pH strongly influences the shape of biochemicals by altering the proportion of dissociation of acidic groups on the molecule. Large biomolecules may have numerous acidic groups with various pKa values. Varying the pH adds or removes hydrogen ions. This affects which portion of the molecule can form hydrogen bonds, or changes the polarity of a molecule, thereby significantly changing its shape and function. TABLE 3.2 pKa Values for Some Biologically Important Chemicals Compound pKa Phosphoric acid (pK1 ) Citric acid (pK1 ) Formic acid Lactic acid Benzoic acid Acetic acid Citric acid (pK2 ) 2.0 3.1 3.8 3.9 4.2 4.7 4.7 Compound Carbonic acid (pK1 ) Citric acid (pK3 ) Phosphoric acid (pK2 ) Boric acid Ammonium Carbonic acid (pK2 ) Phosphoric acid (pK3 ) pKa 6.3 6.4 6.7 9.2 9.3 10.4 12.4 40 THE SUBSTANCES OF LIFE 3.4 PHYSICOCHEMICAL INTERACTIONS Atoms on a molecule can also be attracted to atoms on another molecule, producing intermolecular forces. These forces are primarily electrostatic. The strength of these forces range from that of hydrogen bonds, the strongest, to the van der Waals forces, the weakest. However, all of them are weaker than the forces involved in chemical bonding. Hydrogen bonds are intermolecular attractions that occur between electronegative atoms on one molecule (e.g., O, N, or Cl) and with hydrogen atoms in another molecule. They will occur only if the hydrogen is bonded to other atoms more electronegative than carbon (e.g., O or N). The latter atoms pull the electrons away from the hydrogen, creating a strong local positive charge. This charge consequently has a strong attraction to electronegative atoms on the other molecules. Even in the absence of polarity-producing asymmetry, a molecule can exhibit a temporary polarity as electrons shift from one side to another, or shift in response to the presence of the electron cloud of another molecule nearby. This produces a weaker electrostatic attraction, the van der Waals force. Attractions due to hydrogen bonds, van der Waals forces, or polarity produce several important effects:  The strength of these attractions allow chemicals to form liquids and solids. In more precise terms: They tend to raise the boiling point and melting point, increase the heat capacity and heat of vaporization, and decrease the vapor pressure.  Dissimilar molecules that have sufficient attraction to each other can dissolve or mix, whereas those that do not attract tend to form separate phases.  They contribute to the shape of large molecules because of attractions between different parts of the same molecule, modified by competition with other attractions, such as with solvent molecules or other dissolved species.  They affect the rate of chemical reactions between like and unlike molecules by affecting the proximity and orientation of molecules to each other. Water illustrates several physicochemical interactions and also plays a key role in biochemistry. Water is the most abundant chemical in living things. Living things are composed of between 50 and 90% water by weight. In humans, 99 of 100 molecules are water. The water molecule is bent; that is, rather than being directly on opposite sides of the oxygen, the two H

O bonds form a 104.5 angle with each other. The electronegativity of the oxygen pulls the electrons from the hydrogen. This leaves the molecule with a strong dipole, and each atom can form hydrogen bonds with other water molecules. These properties give it strong self-attraction. This results in the highest boiling point and heat of vaporization of any chemical of similar size or molar mass (i.e., molecular weight). Water’s heat capacity and latent heat helps it regulate the temperature of living things, especially animals, which can generate heat at a rapid rate. Evaporation of water carries away a large amount of heat. If a human consuming 2000 Calories per day (note that 1 dietary Calorie ¼ 1000 calories, distinguished in writing by their capitalization) were to release all that energy as heat, it could be consumed by evaporation of 3418 grams of water, less than 1 gallon. The actual evaporation is less because not all dietary calories are released as heat, and some heat is lost by convection. Water’s polarity also enables it to reduce the repulsive forces between charged particles such as ions, because it can align itself with the ion’s electric field. The reduction in OPTICAL ISOMERS TABLE 3.3 Substance Water Methanol Acetone Chloroform Benzene 41 Properties of Various Chemicals Compared to Water Molar Mass (g/mol) Boiling Point ( C) 18 32 58.1 119.5 78 100 65 56.2 61.7 80.1 Specific Heat Heat of Dielectric Vaporization (cal/g) Strength 1.0 0.6 0.51 0.24 0.5 585 (20 C) 289 (0 C) 125 (56 C) 59 (61 C) 94 (80 C) 80 33 21.4 5.1 2.3 Source: Smith et al. (1983), Table 2.2. electrostatic force relative to a vacuum is called the dielectric strength. This property is what makes water an excellent solvent for ions, since less work is required to place the ion into the water matrix. In addition, the polarity and hydrogen-bonding capability of water facilitates direct attraction to ions and other molecules that are polar or form hydrogen bonds. Polar or hydrogen-bonding compounds with appreciable solubility in water are termed hydrophilic (water-loving). Nonpolar compounds with negligible aqueous solubility are called hydrophobic, or lipophilic. Hydrophobic compounds are important for forming separate phases within cells. The resulting interfaces are an important location for many biochemical reactions and control the movement of hydrophobic toxicants into and out of cells. Table 3.3 summarizes some of the interesting physicochemical properties of water and compares them to other solvents. The physicochemical forces associated with large biological molecules depend in a very complex way on the complete chemical structure of the molecule and on its chemical environment. However, much of their behavior can be described in terms of local chemical structures, called functional groups, some of the most important of which are listed in Table 3.4. Consider any hydrocarbon molecule, consisting only of a carbon backbone and hydrogens. Replace any of the hydrogens with one of the functional groups from Table 3.4 and you produce a compound with very different properties. A simple example is methane, CH4. Replace a hydrogen with a hydroxyl group, and methanol results, which has a much higher boiling point and forms a liquid that is completely miscible with water. Replacing with carboxyl forms acetic acid. Taking the three-carbon hydrocarbon propane and replace one hydrogen from each carbon with a hydroxyl produces glycerol, a basic component of biological fats. 3.5 OPTICAL ISOMERS One of the simplest of the sugars is glyceraldehyde, which has two isomeric forms: CHO H C OH CH2OH d(+)-glyceraldehyde CHO HO C H CH2OH l(−)-glyceraldehyde These structures may seem practically identical, and in fact they have the same physicochemical properties, such as melting point and aqueous solubility. However, the difference between them is biochemically critical. One is an optical isomer of the other; that 42 THE SUBSTANCES OF LIFE TABLE 3.4 Functional Groups and Their Propertiesa Hydroxyl (OH) Polar; increases solubility in water; forms hydrogen bonds; characteristic of alcohols Polar; most important acidic group in biochemicals; pKa depends on R (the more electronegative, the lower the pKa ) R OH O Carboxyl (COOH) R C OH Amine (NH2) R NH2 Aldehyde (CHO) R C O H Keto (CO) R1 Weak base; electronegative nitrogen attracts an additional Hþ from solution to become positively charged; common in proteins Polar and water soluble; common in sugars and as fermentation products O Polar and water soluble; common in sugars and as fermentation products H Nonpolar; reduces water solubility C R2 Methyl (CH3) R C H H OH Phosphate (PO4) Polar acidic group; important in energy metabolism; found in DNA, sugars; additional organic side chains can replace the two hydrogens; phosphorus is usually present only in the form of phosphate R P O OH Sulfhydryl Disulfide a R S R1 S Polar; forms disulfide bonds to link molecules H S R2 Formed from two sulfhydryl bonds; important in protein folding R stands for the rest of the molecule. is, they rotate polarized light in opposite directions due to their mirror-image asymmetry. To understand this, it is necessary first to recognize that the four bonds that a carbon atom can participate in are arranged to point to the vertices of a tetrahedron if each bond connects to identical structures. This can be seen in Figure 3.4, which shows the tetrahedral H C H H H (a) (b) Figure 3.4 (a) Tetrahedral structure of carbon bonding and (b) a methane molecule. (Based on Gaudy and Gaudy, 1988.) OPTICAL ISOMERS 43 structure and a methane molecule having that form. If the functional groups on all four bonds are identical, the angle between any two bonds will be 109 . The difference between the two types of glyceraldehyde is not obvious in the twodimensional representation above. It seems that it should be possible to rotate the molecule around two of the bonds to change one form to the other. That it is not possible will be clearer if the molecule is viewed in its true three-dimensional form. The central carbon atom could be viewed as being at the center of a tetrahedron, with each of its four bonds pointing to a vertex. However, each of these bonds connects to a different group: an H, an OH, a CHO, and a CH2OH. Such asymmetrical carbons are called chiral centers. Molecules with chiral centers can rotate polarized light either to the right, designated (þ), or the left, designated (
). Biochemical compounds are often designated d- for dextro, meaning ‘‘right’’ or l- for levo, meaning ‘‘left,’’ based on a relationship to the structure of (þ)glyceraldehydes or (
)glyceraldehydes, respectively. More complex molecules may have multiple carbon atoms that can form centers for optical rotation. In such cases, the d- or l- notation indicates a relationship to the structure of glyceraldehydes, not whether the molecule actually rotates light to the right or left. Whether the molecule actually rotates polarized light to the right or left is designated by including (þ) or (
) in the name, respectively. Figure 3.5 shows three-dimensional views of d- and l-glyceraldehyde. In part (a) the two forms seem identical, but a close examination will show that bonds that point out of the plane of the page in one form point inward in the other. The difference between the two becomes more clear in part (b), which could be formed by taking hold of the hydrogen bonded to the central carbon of each compound and pointing it toward the viewer. The CH2OH group is pointed to the left (the oxygen is hidden behind the carbon). Then it can clearly be seen that the two compounds cannot be superimposed on each other Figure 3.5 Three-dimensional views of the glyceraldehyde structure: (a) mirror-image views; (b) views showing the orientation with hydrogen of the central carbon pointed toward the viewer. 44 THE SUBSTANCES OF LIFE because the three groups attached to the central carbon atom are arranged in opposite directions around the carbon. The importance of chirality is that these isomers are biochemically different and will react differently from each other. Fundamentally, optical isomers have different shapes from each other. The shape of a molecule determines how it can form complexes with other molecules. As described below, the formation of complexes, especially with enzyme molecules, is a key step in a biochemical reaction. For example, d-glucose can be used for energy by the brain, but l-glucose cannot. Similar differences are true for many other stereoisomeric biochemical compounds. Another name for d-glucose is dextrose, the familiar ingredient in manufactured food items. 3.6 THE COMPOSITION OF LIVING THINGS Four groups of compounds are of primary importance in living things: carbohydrates (including sugars, starches, cellulose, and glycogen), lipids (fats and oils), proteins, and nucleic acids (which form DNA and RNA). The first three of these form the majority of cell dry weight and are important for structural material, energy metabolism, and other metabolic functions. Nucleic acids are significant in reproduction and in energy metabolism. Finally, there are many compounds that do not fit neatly into these categories or may be hybrids of two or more. 3.6.1 Carbohydrates Carbohydrates include sugars, starches, and structural materials such as cellulose. All have the empirical chemical formula (CH2O)n. For example, glucose is C6H12O6, so n is 6. Glyceraldehyde is one of the simplest carbohydrates, with an n of 3. The large number of hydroxide groups on carbohydrates renders them hydrophilic. Carbohydrates are classified into several groups: Monosaccharides are the simplest and are building blocks for the others. They have relatively low molar masses, and n in the formula can range from 3 to 9. Monosaccharides can form chains, called polymers, producing disaccharides, which are formed from pairs of monosaccharides, or the long-chain polysaccharides, which can have molar masses as high as 1 million. Large molecules such as polysaccharides, proteins, or DNA are called macromolecules. The structures of several monosaccharides that are commonly found in nature are shown below. Ribose is a five-carbon sugar; the others are six-carbon sugars. Glucose is a particularly important six-carbon sugar. It is the principal sugar formed by photosynthesis and is the main immediate source of energy for all cells. Nervous tissue in animals can only use glucose for energy. H C O HO C H H C O H C OH CH2OH H C O H C OH C O HO C H H C OH HO C H H C OH H C OH HO C H H C OH H C OH CH2OH H C OH H C OH H C OH CH2OH CH2OH d(+)-glucose d(+)-galactose CH2OH d(+)-ribose d(−)- fructose THE COMPOSITION OF LIVING THINGS 45 One end of monosaccharides such as glucose reacts spontaneously with either the other end or the adjacent carbon to form ring structures. For example, glucose can form a sixmembered ring, and fructose, a five-membered ring. CH2OH OH CH2OH OH O OH CH2OH O OH OH OH OH d-glucose d-fructose The rings are closed by an oxygen atom. The ring form and the open-chain form freely interconvert. However, for glucose, for example, the equilibrium highly favors the ring form. A number of monosaccharide derivatives are important. Conversion of ends to

COOH groups produces the sugar acids, such as glucuronic acid. Fermentation may result in the production of sugar acids as intermediates. Amino sugars are formed by replacing one of the hydroxyl groups on certain monosaccharides with a nitrogen-containing amino group (see below under proteins). Another derivative is the deoxy sugar, formed by replacing one of the hydroxy groups with a hydrogen; thus, one of the carbons will have two hydrogens. A very important deoxy sugar is deoxyribose, an important component of DNA. Monosaccharides can also form a special type of bond with each other or with other molecules. This glycosidic bond forms between a hydroxide on the monosaccharide and a hydroxide on another molecule, which may be another monosaccharide, with the elimination of one molecule of water. Again, the link will be through an oxygen atom. If the other molecule is another monosaccharide, the result is a disaccharide. Familiar table sugar is sucrose, a disaccharide formed from the monosaccharides glucose and fructose. Lactose, the sugar in milk, is formed from glucose and galactose. The same two monosaccharides can form disaccharides in several ways, depending on orientation and location of the connection. For example, maltose and cellobiose are both made from two glucose residues. CH2OH OH CH2OH OH O OH O O OH OH CH2OH OH sucrose Glycosidic bonds can form longer-chain carbohydrates called oligosaccharides, those with only a few monosaccharides, which include the disaccharides, and the much longerchain polysaccharides. Starch is a type of polysaccharide produced by plants for energy storage. Humans obtain most of their dietary carbohydrate in the form of starch from grain. Starch is a chain of glucose residues bonded as in maltose. The chain may be straight, as in amylose, which makes up about 20% of potato starch; or it may be branched, as in amylopectin, which forms the other 80% of potato starch. Animals store carbohydrates in a polysaccharide called glycogen. Glycogen is similar to starch except that the chain is much more highly branched. It is thought that by having more ‘‘ends’’ to the glycogen molecule, it is more available for rapid conversion to 46 THE SUBSTANCES OF LIFE glucose for the sudden energy demands of animals. Depletion of glycogen in the muscle may cause the ‘‘wall’’ experienced by marathoners after several hours of running, which prevents them from continuing the race. With glycogen gone, the body switches to fat, which does not provide energy fast enough and produces other physiological stresses. In mammals, skeletal muscle contains about two-thirds of the body’s glycogen, and the liver holds most of the rest. The liver uses the glycogen to control glucose levels in the blood. Figure 3.6a shows the basic structure common to both starch and glycogen. Cellulose is an unbranched polysaccharide also composed exclusively of glucose residues, but with an important difference from starch. The glycosidic bond is reversed, as in the cellobiose disaccharide (see Figure 3.6b). This prevents the molecule from twisting, making it stiffer. As a result, it finds use as a structural material located in the cell walls of plants and forming the major component of wood. (It should be noted that the hardness of wood comes not from cellulose but from lignin, described below.) Another important fact about cellulose is that only a very few animals (such as garden snails) can digest it. Most cannot break it down to glucose to make the stored energy available. However, several animals, such as termites and cows, have developed associations with microorganisms that live in their digestive systems. The microorganisms accomplish the digestion for the animals. CH2OH O (a) CH2OH O O O CH2OH CH2OH O CH2 O O CH2OH O O CH2OH O O O O (b) CH2OH CH2OH O CH2OH O O CH2OH O O CH2OH O O O O Figure 3.6 Polymers of glucose: (a) starch or glycogen showing a maltose repeating disaccharide unit; (b) cellulose with a cellobiose repeating unit. THE COMPOSITION OF LIVING THINGS 47 Another structural polysaccharide is chitin, which forms the hard shell of arthropods such as insects and crabs, as well as the cell walls of most fungi. Chitin is a polymer of a sugar amine, N-acetyl glucosamine; crustaceans also include calcium carbonate in their shells. Chitin has been studied for use as an adsorption media for removing heavy metals from water. Other important structural polysaccharides include agar and carrageenan, which are extracted from seaweed. The former is used as a substrate for culturing bacteria, and the latter is used as a food thickener. 3.6.2 Lipids Lipids refer to a loose category of compounds with the common property that they have fairly low solubility in water or are extracted from biological materials by solvents having polarity much less than water, such as ethanol or chloroform. There are five major types, of which the first four are described here and the fifth in a later section.      Fatty acids, long-chain aliphatic carboxylic acids Fats, esters of fatty acids with glycerol Phospholipids, esters of phosphate and fatty acids with glycerol Lipids not containing glycerol, including waxes and steroids Hybrid lipids, such as those combined with carbohydrates or proteins Fatty acids are simply straight-chain hydrocarbons with a carboxylic acid functional group at one end (Figure 3.7). They usually, but not always, have an even number of carbon atoms. It is the hydrocarbon chain that imparts hydrophobicity, since the carboxyclic acid group is water soluble. The larger the chain is, the more hydrophobic the molecule. The simplest fatty acid is formic acid, where a simple hydrogen makes up the variable R-group. The most familiar is acetic acid, formed with a methyl group. Vinegar is about 5% acetic acid. Both of these are quite water soluble. The melting point of fatty acids tends to increase with chain length. Fatty acids usually do not accumulate in nature. Systems may be engineered to produce them, as is done in fermentation processes. An important environmental application is anaerobic digestion, in which fatty acids consisting mainly of acetic acid, but also Basic structure: R— COOH Saturated fatty acids: Formic HCOOH Acetic CH3COOH Proprionic CH3CH2COOH n-Butyric CH3(CH2)2COOH Caproic CH3(CH2)4COOH Palmitic CH3(CH2)14COOH Stearic CH3(CH2)16COOH Unsaturated fatty acids Oleic CH3(CH2)7CH=CH(CH2)7COOH Linoleic CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH Arachidonic CH3(CH2)4-(CH=CH-CH2)4-(CH2)2-COOH Figure 3.7 Structures of some of the more common fatty acids found in nature. 48 THE SUBSTANCES OF LIFE O H2C H2C OH HC OH H2C OH O 3 R O O + C C R HC O C R + 3H2O O HO H2C O C R Figure 3.8 Formation of triglyceride from glycerol and fatty acids. proprionic, butyric, and others, can accumulate to levels typically below 1%, as an intermediate in methane production. Saturated fatty acids are those in which the maximum number of hydrogens have been bonded with the carbons in the hydrocarbon chain. If a hydrogen is removed at a point in the chain, the corresponding carbon has an extra bond to form, to fill its complement of four. It satisfies this requirement by forming a double-bond with its neighbor (which also loses a hydrogen). Fatty acids containing one or more double-bonded carbons are called unsaturated fatty acids. Unsaturation puts a kink in the chain, reducing the ability of the molecules to pack together. As a result, they are less likely to form solids (i.e., their melting points are lowered). Except for formic acid (pKa ¼ 3:75), all of the saturated fatty acids have acid dissociation constants averaging around 4.85, similar to that of acetic acid (pKa ¼ 4:76). Fats are formed from the covalent bonding of three fatty acids with the three hydroxides of a glycerol (Figure 3.8). For this reason they are often called triglycerides. The bonding of a hydroxyl of an alcohol with the hydroxyl of a carboxylic acid, with elimination of a water molecule, results in an ester linkage. This eliminates the hydroxide from the alcohol and the ionizable portion of the acid. With the loss of these functional groups, polarity is greatly reduced, ionization is eliminated, and water solubility is therefore decreased. Because the ester linkage is only slightly polar, the properties of fats are dominated by the properties of the hydrocarbon chains of the fatty acids that form them. The reverse of the esterification reaction is hydrolysis, which means ‘‘splitting with water.’’ Hydrolysis of ester bonds is catalyzed by Hþ or by enzymes called lipases. The ester bond can also be hydrolyzed in a strong basic solution, in a reaction called saponification. The salts of the fatty acids thus formed are soaps. In fact, soap was made in preindustrial households by reacting fats collected from meat with lye (sodium hydroxide) leached from ashes. Oils are fats that are liquids at room temperature. Fats made from unsaturated fatty acids are unsaturated fats. As with the fatty acids, they will have lower melting points. This is why you may read that margarine or shortening contains ‘‘partially hydrogenated fats.’’ Fats are hydrogenated by reacting unsaturated vegetable oils with hydrogen, reducing the number of double bonds, in order to decrease the melting point, allowing the oil to form a solid. In a similar way, living things select for the saturation of fats to obtain properties that are useful. The major functions of fats in organisms are for energy storage and structural use. Fats yield twice as much chemical energy as carbohydrates or proteins; fats average about 9.3 Calories per gram vs. 4.1 Calories per gram for both proteins and carbohydrates. (Note that the dietary Calorie, which must be capitalized, is equivalent to the kilocalorie THE COMPOSITION OF LIVING THINGS TABLE 3.5 49 Percentage of Types of Fats in Some Dietary Fats and Oils Saturated Monounsaturated Polyunsaturated Other Cholesterol (mg/tB) 6 9 14 51 54 77 62 12 77 44 30 6 31 78 9 4 4 2 1 1 0 1 12 15 0 0 0 14 33 0 Canola oil Safflower oil Olive oil Beef fat Butter Coconut oil commonly used in thermodynamics.) However, fat storage is long term. The energy contained in fats is not released as rapidly as with the polysaccharides starch or glycogen. Other functions of fats are for insulation for animals exposed to cold, and for flotation in marine animals. The blubber of sea mammals is an example of both of these functions. The degree of fat saturation in the human diet is linked to human health. A small amount of unsaturated fat is required in the diet, because humans cannot form fatty acids with double bonds. One of these in particular, linoleic acid, can be converted into all the other fatty acids needed by humans. For this reason, it and several others are placed in a dietary group called the essential fatty acids. A mammalian diet containing a high proportion of saturated fats is associated with cardiovascular disease, via its connection with cholesterol and blood lipoproteins. (Cholesterol is another type of lipid, described below. Lipoproteins are hybrid compounds, also described below.) Among the unsaturated fats, monounsaturated fats, those with only one double bond, seem to be even more healthful than polyunsaturated fats, those with more than one double bond. Table 3.5 shows how various fats compare in these dietary constituents. Olive oil has the highest monounsaturated fat content of all. This has lent support to the ‘‘Mediterranean diet,’’ in which olive oil replaces butter and other animal fats in recipes, to the extent of placing a plate of oil on the table for dipping bread instead of a butter dish. Lest one should think that only animal fats are suspect, notice the high saturated fat content of coconut oil. High polyunsaturated fat content also seems to beneficially lower blood lipid concentration. Safflower oil is best in this regard, followed by corn, peanut, and cottonseed oils. Phospholipids are particularly critical to life, as they form the basic structure of cell membranes. Glycerol can form ester linkages with inorganic as well as organic acids. Phospholipids consist of ester linkages of glycerol with two fatty acids and with phosphoric acid in the third position. Usually, another of the phosphate hydroxyl groups form, in turn, another ester bond with still another organic molecule, called a variable group. The variable group often contains nitrogen, adding to the polar character of the lipid. OH R O P H O C H HC O C O H2C O C O O phospholipid structure 50 THE SUBSTANCES OF LIFE Polar head Nonpolar tail Phospholipid Monolayer Monomers (a) Micelle (b) Figure 3.9 Surfactant structures formed in solution: (a) behavior of surfactants in solution; (b) phospholipid bilayer structure. This structure results in a surfactant, a molecule that has a hydrophilic part and a hydrophobic part. Similar to soaps and detergents, if there are enough molecules in solution, they will form aggregates called micelles, a sort of ‘‘circling the wagons’’ in which the polar ends face the water and the hydrophobic ends form a separate phase in the interior of the micelle (Figure 3.9). One of the phospholipids is lecithin, which is important in metabolism of fats by the liver. Egg yolks are rich in lecithin, and its detergent nature helps maintain the emulsion between oil and vinegar in mayonnaise. Biological lipids show an important difference in behavior from other surfactants. Under the right conditions, they form micelles with multilayered structures that can become vesicles (water-filled cavities) bounded by a lipid bilayer, as also shown in Figure 3.9. This important structure is the basis for the cell membrane, and therefore for the cell itself. The bilayer membrane forms a barrier to the uncontrolled passage of water-soluble constituents into and out of the cell. However, its lipophilicity makes it the site of action of many lipophilic pollutants, such as some industrial organic solvents. Other lipids: Since the definition of lipids is based on physicochemical properties and not chemical structure, it is not surprising that the group is very diverse. A variety of other biological compounds fit the category. Waxes are long-chain fatty acids combined with long-chain alcohols other than glycerol. They are formed by plants to produce protective and water-conserving layers on their surfaces and by animals such as the honeybee, or for ear canal protection. Terpenes are long-chain hydrocarbons based on units similar to the compound isoprene. They include essential oils from plants, among other compounds. One important group of compounds that is in this catch-all class of lipids is the steroids. Steroids consist of four fused rings, three with six carbons and one with five, and with a hydroxide at one end and a hydrophobic ‘‘tail’’ at the other. Steroids are important regulatory chemicals in plants and animals. They are able to move through cell membranes formed by lipid bilayers. The sex hormones estrogen and testosterone are steroids, as are the fat-soluble vitamins A, D, and E. The most abundant steroid in animals is cholesterol. It forms an essential part of the cell membrane, affecting its fluidity. However, factors THE COMPOSITION OF LIVING THINGS 51 related to diet and heredity can cause cholesterol to form obstructive deposits in blood vessels, causing strokes or heart attacks. HO cholesterol Another lipid that is important in biological wastewater treatment is poly-b-hydroxybutyric acid (PHB). This is used for energy storage by some bacteria. O C CH3 O O CH C H2 C CH3 O O CH C H2 C O poly-b-hydroxybutyric acid 3.6.3 Proteins Proteins have a central role in cell function. Like carbohydrates and lipids, they are involved in structure and in energy metabolism. More important is the function unique to proteins: Each of the thousands of enzymes in living things, each of which catalyzes a specific biochemical reaction, is a protein specialized for that task. In addition, they may act as biochemical regulators or hormones, such as insulin; transport chemicals such as hemoglobin, which transports oxygen in the blood; or they may be responsible for motility, as in the cilia and flagella of protists. Proteins are composed of one or more chains of amino acid repeating units. Each individual chain is called a polypeptide. In turn, amino acids are compounds in which a central carbon is covalently bonded to three functional groups: an amine, a carboxylic acid, and a variable organic side group: COOH R C H NH2 Many amino acids are found in nature, but only 20 are commonly part of proteins. In contrast to polysaccharides, proteins are formed not from one or two repeating units, but from all 20, and in any sequence. This makes possible a virtually unlimited variety of structure and of corresponding function. A chain of n amino acids can form 20n different proteins. For a length of only five acids, this gives 3.2 million combinations. Typical chain lengths are from 100 to several thousand. Of course, only certain combinations actually occur. The human body has about 100,000 different proteins. Table 3.6 shows the 20 amino acids commonly found in proteins. Some have simple side groups (e.g., glycine and alanine), others are complex (e.g., tryptophan). Two contain sulfur (cysteine and methionine). Some have ionizable side groups, such as the carboxylic 52 THE SUBSTANCES OF LIFE TABLE 3.6 Amino Acids Commonly Found in Proteins and Their pI Values Name Symbol Structure pI Amino Acids with Nonpolar R Groups H Alanine Ala H3C C COO− 6.00 NH3+ Isoleucinea Ile Leucinea Leu H H3C CH2 CH C COO− H3C NH3+ H3C 6.02 H CH CH2 C COO− H3C NH3+ H3C S CH2 CH2 C COO− 5.98 H Methioninea Met 5.74 NH3+ H Phenylalaninea CH2 C COO− Phe 5.48 NH3+ Proline Pro H2C CH2 H C H2C NH + COO− 2 6.30 H Tryptophana Trp Valinea Val C CH2 H3C H3C CH N H H C COO− NH3+ CH C COO− 5.89 5.96 NH3+ Amino Acids with Uncharged Polar R Groups O Asparagine Asn H2N H C C COO− 5.41 NH3+ H Cysteine Cys HS CH2 C COO− 5.07 NH3+ O Glutamine Gln H2N H C CH2 CH2 C COO− NH3+ 5.65 53 THE COMPOSITION OF LIVING THINGS TABLE 3.6 ðContinuedÞ Name Symbol Structure pI H Glycine Gly H C COO− 5.97 NH3+ H Serine Ser HO CH2 C COO− 5.68 NH3+ H Threonine a Thr H3C CH C COO− 5.60 OH NH3+ H Tyrosine Tyr CH2 C COO− HO 5.66 NH3+ Amino Acids with Acid R Groups (Negatively Charged at pH 6.0) −O Aspartic acid Asp H C CH2 C COO− O −O Glutamic acid Glu 2.77 NH3+ H C CH2 CH2 C COO− O 3.22 NH3+ Amino Acids with Basic R Groups (Positively Charged at pH 6.0) H Arginine Arg + H3N C NH CH2 CH2 CH2 C COO− 10.76 NH3+ NH H C CH2 C COO− HC Histidine His +HN NH 7.59 NH3+ C H H Lysine a Lys +H N 3 CH2 CH2 CH2 CH2 C COO− NH3+ a Essential amino acids. 9.74 54 THE SUBSTANCES OF LIFE acids in aspartic acid and glutamic acid, or the basic amines of lysine, histidine or arginine. Amines, including the one connected to the central carbon, ionize by accepting an additional hydrogen ion at pH levels below a characteristic pK value. Because all amino acids have both an acidic and a basic functional group, all will have an ionic charge at either sufficiently high or low pH values. For some, it is possible at certain pH values for both groups to be ionized: COO− COOH R C H R C H NH3+ NH2 These behaviors are important in forming attractions between different parts of the protein molecule, affecting its shape. Table 3.6 also gives the isoelectric point (pI), which is the pH at which the number of positive charges on the amino acid in solution equals the number of negative charges. The fact that all amino acids contain nitrogen explains the importance of that element to all organisms. Nitrogen limitations can cause growth problems in biological wastewater treatment and in natural ecosystems. Humans also have a nutritional requirement for proteins. We can form many of the amino acids from other compounds. However, we cannot synthesize the following eight, which are called essential amino acids: isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. These must be provided in the diet daily, since free amino acids are not stored in the body. The absence of any one causes protein synthesis to stop. Most animal proteins contain all eight, but many plant proteins do not, or do not have them in balanced amounts. For this reason vegetarians need to be aware that they must eat vegetables in combinations that eliminate deficiencies. For example, rice is deficient in lysine but has sufficient methionine, whereas beans are low in methionine but contain adequate lysine. However, beans and rice together in a diet supply a complete protein supply. Amino acids form polypeptides by covalently bonding the carbon from the carboxylic acid group to the nitrogen of the amine, with loss of a water molecule. The result is called a peptide bond: H R1 O C C NH2 OH H + R2 NH2 OH COOH C C N C R2 + H2O H O O C C R1 NH2 H H peptide bond formation Because of the amino acid functional groups, proteins also possess charges in solution, which vary with pH. Like individual amino acids, proteins have an isoelectric point. These can range from less than 1.0 for pepsin, the digestion enzyme that must act under acidic conditions in the stomach, to 10.6 for cytochrome c, which is involved in cellular respiration. Unlike polysaccharides, which can have random lengths and random branching, each peptide is a single chain with a precise sequence of amino acids. Changing even a single amino acid can destroy the ability of the resulting protein to perform its function. Furthermore, the peptide chain must arrange itself into a complex shape, which is determined by the exact amino acid sequence, and often by the method by which the cell machinery THE COMPOSITION OF LIVING THINGS Ser Gly Leu Val Glu Glu Cys S S Cys Leu 55 Tyr Glu Val Leu Ser Glu Cys Ala Asp S S Val Asp Glu His Leu Tyr Cys Cys Gly Asp S S Ser His Tyr Leu Val Cys Gly Glu Leu Leu Val Glu Arg Gly Ala Phe Phe Thr Tyr Ala Lys Pro Figure 3.10 Tertiary and quarternary protein structure as shown in bovine insulin. This protein consists of two polypeptide chains joined by two disulfide bonds. Another disulfide bond within the smaller chain contributes to the molecule’s shape. (Based on Bailey and Ollis, 1986.) constructs the protein. The structure of a protein has three or four levels of organization. The primary level is the actual amino acid sequence. The secondary level refers to relatively local arrangements such as coiling into a helix or folding into a pleated sheet. The helix is held together by hydrogen bonds between the peptide bonds of every fourth amino acid. The tertiary level of organization is larger-scale folding and coiling, to give the overall shape to the molecule. Some proteins will exhibit the quaternary level of structure, in which several polypeptides are linked together by a variety of attractions, including hydrogen bonds, ionic attraction, or covalent disulfide linkage between cysteine amino acids on the two peptides. Hemoglobin, for example, consists of four polypeptide units. Figure 3.10 shows an example of protein structure. If the protein forms a compact, watersoluble state, which the majority of proteins do, they are called globular proteins. Fibrous proteins are elongated and often function in structural applications in connective tissue, contractile tissue, or as part of the hair or skin in mammals. Proteins molecules often have other chemical compounds, called prosthetic groups, included in their structure, usually through noncovalent bonding. Often, they include metal ions. Hemoglobin contains four organic prosthetic groups, each containing an iron atom. Other proteins may contain chromium, copper, or zinc, for example. This is one of the reasons that humans and other organisms have a nutritional requirement for some heavy metals. 56 THE SUBSTANCES OF LIFE Since the higher levels of protein structure depend on relatively weak bonds such as hydrogen bonds, they are easily disrupted by increasing temperature or by changing pH or ionic strength. Such changes may result in conversion of the protein to a nonfunctional form, which is said to be denatured. These changes are often reversible. For example, hair can be curled by wrapping it around a rod and heating. This breaks hydrogen bonds, which re-form upon cooling, ‘‘freezing’’ the protein in the new shape. However, there is tension in the hair fibers, and with time the hydrogen bonds gradually rearrange into their former relationship, losing the curl. A ‘‘permanent’’ rearrangement can be made by using chemical treatment, which breaks disulfide bonds between cysteine residues in hair proteins, then re-forms them in the curled shape. A common example of irreversibly denaturing proteins by heat is the cooking of eggs. Heat disrupts the globular albumin proteins, which do not return to their native state upon cooling. Enzymes are protein catalysts that increase biochemical reaction rates by factors ranging from 106 to 1012 over the uncatalyzed reactions. They often include nonamino acid portions that may be organic or consist of metallic ions. These are called cofactors. Most enzymes are named with the suffix -ase. For example, lipase is an enzyme that digests lipids. Another enzyme is lactase, which catalyzes the breakdown of milk sugar, the disaccharide lactose, into monosaccharides glucose and galactose. Many adults, and almost all non-Caucasian adults, lose their ability to produce lactase after early childhood. However, some bacteria, including Escherichia coli, produce a different lactose-digesting enzyme. Adults lacking lactase who eat milk products have abdominal disturbances when the bacteria in the gut begin to produce gas using the lactose. Enzymes are very specific; each catalyzes one or only a few different reactions, which is sensitively controlled by its shape. It is remarkable that contrary to reactions in aqueous media in the laboratory, enzyme-catalyzed reactions produce few side reactions. Equally remarkable is the fact that, with enzymes, a wide variety of reactions are promoted at mild conditions of temperature, pressure, and pH. Each enzyme has at least one active site, the location on the molecule that binds with the substrate(s) (the reactants in the catalyzed reaction). The active site attracts the substrate(s) and holds it, usually by physicochemical forces. Two major mechanisms by which enzymes increase reaction rates are (1) by bringing the reactants close together, and (2) by holding them in an orientation that favors the reaction (Figure 3.11). It is also thought that enzymes can act by inducing strain in specific bonds of bound substrates, making certain reactions favorable. Since the shape of a molecule is so sensitive to its environment, the cell can turn reactions on or off by changing conditions (e.g., pH) or by providing or withdrawing a cofactor or inhibitory compound. Figure 3.12 shows how a cofactor could promote binding of a single substrate with an enzyme. The cofactor binds first with the enzyme, changing the shape of the active site. This allows the substrate to bind, forming the complex. As with all proteins, denaturing stops the function of any enzyme. Enzymes may also require coenzymes, which are molecules that function by accepting by-products of the main reaction, such as hydrogen. Coenzymes differ from cofactors and from enzymes themselves in that they are consumed by the reaction (although they may be regenerated in other reactions). Examples include NAD and FAD, discussed below. Some cofactors and coenzymes cannot be synthesized by mammals and must be included in their diet, making them what we call vitamins. THE COMPOSITION OF LIVING THINGS 57 A Product + B + + Enzyme Enzyme-substrate complex Figure 3.11 Enzyme control of proximity and orientation of substrates. Another important protein function is their use as binding proteins. Hemoglobin is an example of a binding protein that transports oxygen in the blood. Other binding proteins are active in the immune system, which responds to foreign substances in animals. The cell membrane is studded with proteins that function in communicating substances Substrate Substrate + Cofactor + + Enzymesubstratecofactor complex Enzyme Enzyme Figure 3.12 Hypothetical enzyme mechanism involving a cofactor. 58 THE SUBSTANCES OF LIFE and signals into and out of the cell. Cell membrane proteins are also a point of attack for infectious agents such as viruses, or may bind with drugs, leading to reactions that produce their characteristic effects. 3.6.4 Nucleic Acids Nucleic acids do not form a large portion of the mass of living things, but make up for this in importance by being central to reproduction and control of cell function (DNA and RNA) and as the single most important compound in energy metabolism [adenosine triphosphate (ATP)]. DNA and RNA are linear polymers of nucleotides; ATP is a single nucleotide. A nucleotide is a compound consisting of three parts: pyrimidine or purine base þ ribose or deoxyribose sugar þ one or more phosphates The pyrimidines are based on a six-membered ring containing two nitrogens. Only the following three pyrimidines are found in DNA and RNA: thymine, cytosine, and uracil (Figure 3.13). Purines have an additional five-membered ring fused to the pyrimidine. Only two purines are used in DNA and RNA, adenine and guanine. Nucleotides using these bases are labeled with their first letter: A, G, U, C, or T. The five-carbon sugars are bonded to the base, and the phosphate(s) are connected to the sugar via an ester. A nucleoside is the same as a nucleotide, without the phosphate. The nucleoside formed from adenine and ribose is called adenosine. The nucleoside formed from thymine and deoxyribose is called thymidine. Besides forming a chain of phosphates, the phosphate portion of the molecules can form ester bonds to two nucleotide sugars, forming a linear polymer with the phosphates and sugars as a ‘‘backbone’’ and the bases as branches (Figure 3.14). Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are polymers formed from nucleotides. In DNA the sugars are deoxyribose; in RNA the sugars are ribose. Another important difference between these polymers is that DNA does not contain uracil, while RNA never includes thymine. In other words, DNA includes only A, G, C, and T; RNA has only A, G, C, and U. RNA is present only as a single chain. However, two strands of DNA form a fascinating structure called the double helix, discovered by Watson and Crick in 1953. It happens that thymine and adenine on two different strands can form two hydrogen bonds in just the right position relative to each other, and cytosine and guanine on two complementary strands form three such bonds (Figure 3.14). Thus, the sequence of bases on one strand determines the sequence on the other. The two complementary strands are held together by a large number of hydrogen bonds, making the pairing very stable. The resulting structure resembles a ladder, with each sugar–phosphate backbone making one side of the ladder, and the base pairs forming the rungs. A purine is always opposite a pyrimidine, to keep the lengths of the rungs all the same. In addition, the ladder has a right-hand twist to it, making a complete turn every 10 ‘‘rungs’’ or base pairs, producing the famous double helix structure. Notice from Figure 3.14 that the point of attachment at one end of the chain of nucleotides is carbon 5 of the sugar, and at the other end it is carbon 3. This gives a direction to 59 THE COMPOSITION OF LIVING THINGS PURINES PYRIMIDINES NH2 O N H3C N NH N OH N N O P O CH2 O O OH O OH 5' 4' P O CH2 O 1' OH H 3' 2' OH OH Adenine Thymine O NH2 N NH N N OH N NH2 N O P O OH CH2 O O O OH P O CH2 O OH OH Guanine OH Cytosine O NH N OH O P O CH2 O O OH OH OH Uracil Figure 3.13 Nucleotide structures. the chain. The ends are labeled 30 and 50 . You can also see that the complementary DNA chains run in opposite directions. The importance of DNA is that it carries all of the genetic information of all living things. The information is coded in the sequence of bases in the DNA molecule. The two strands, although different, are complementary and carry the same information. That 60 THE SUBSTANCES OF LIFE 3' end CH3 5' end O O H O O H N P N N OH H N N 5' N O O 4' 1' 3' CH2 Thymine N CH2 H O O O Adenine HO 2' O O H O P N H O P O OH N N H N O N O CH2 O H N CH2 N O Cytosine O N H HO P Guanine O O 5' end O 3' end Figure 3.14 DNA molecule section showing the phosphate-sugar ‘‘backbone.’’ is, if one strand has the bases ATGCCACTA, the other strand must be TACGGTGAT, to form the following pairings: 30 . . . ATGCCACTA . . . 50 50 . . . TACGGTGAT . . . 30 What does DNA code for? Very simply, by specifying the sequence of amino acids, it contains instructions for the construction of all the proteins that the organism can make. Recall that proteins can have 20 different amino acids. So how can four nucleotide bases code for all 20? The answer is that the DNA bases code in groups of three. THE COMPOSITION OF LIVING THINGS 61 The sequence for the bottom strand in the example above, and the corresponding amino acid sequence, is DNA : AA : TAC
methionine
GGT proline

GAT leucine As mentioned above, RNA uses uracil in place of thymine and ribose sugar instead of deoxyribose. In addition, it is a single strand. The major function of RNA is to communicate the DNA code from the cell nucleus to the cytoplasm, where proteins are synthesized. More details on the mechanisms involved are provided in Section 6.2.1. RNA has another function, recently discovered. It can act as a catalyst, similar to protein enzymes. RNA with this capability are called ribozymes. One school of thought holds that because RNA can act as both a genetic template and as a catalyst, it may be that when life originated, it was based on RNA for both of those functions. Several nucleotide monomers are important participants in biochemical reactions. More is said in Section 5.1.3 about adenosine triphosphate (ATP) and its central role in energy metabolism. The cell uses a number of other nucleotides. Cyclic adenosine monophosphate (cAMP) is involved in regulation of cell metabolism. Adenine is combined with other organic molecules to form a number of coenzymes, including: flavin adenine dinucleotide (FAD) nicotinamide adenine dinucleotide (NAD) nicotinamide adenine dinucleotide phosphate (NADP) guanosine triphosphate (GTP) These compounds are important in the mechanisms for many biochemical processes, including photosynthesis and respiration, as discussed below. 3.6.5 Hybrid and Other Compounds Hybrid compounds are those composed of a combination of two or more types of compounds, such as sugar combined with protein or with lipid. Some have already been discussed; examples are some proteins with their prosthetic groups; the nucleotides themselves, which contain sugars; and the nucleic acid coenzymes. In addition, sugars commonly combine covalently with lipids and proteins. Many are important in cell membranes. For example, peptidoglycans have the interesting property that they form a two-dimensional polymer (covalently bonded in both the x and y directions) so that they encapsulate bacterial cells with a single huge macromolecule. Lipopolysaccharides include bacterial cell membrane components called endotoxins, which are responsible for powerful toxic effects in animals. Bacteria important in wastewater treatment secrete a coating of polysaccharides and lipopolysaccharides that enables them to flocculate into large aggregates or to form slime layers called biofilms. This improves their ability to capture particulate food matter. Lipoproteins are noncovalently bound lipids and proteins. Since lipids are insoluble in water, they are transported in the blood by being associated with proteins in this way. In other words, proteins act as a ‘‘detergent’’ to solubilize lipids, including cholesterol. Despite cholesterol’s bad reputation related to disease of the circulatory system, it is an essential component of animal cell membranes and a precursor for steroid hormones and 62 THE SUBSTANCES OF LIFE bile acids (which aid in lipid digestion). However, people with high levels of cholesterol in the blood tend to have higher incidences of arteriosclerosis, the narrowing and blocking of arteries by cholesterol deposits. This can lead to strokes and heart attacks. Blood lipoproteins can be separated into fractions distinguished by density. One of the fractions is low-density lipoprotein (LDL). Blood cholesterol is concentrated in the LDL fraction, resulting in its being labeled as ‘‘bad’’ cholesterol. A high LDL level is associated with the intake of saturated fats and alleviated by the intake of monounsaturated fats. Lignin is a polymer of various aromatic subunits, such as phenylpropane. It is produced by woody plants as a resin, bonding the cellulose fibers into a tough composite. 3.7 DETECTION AND PURIFICATION OF BIOCHEMICAL COMPOUNDS The tremendous amount that is known about biochemistry may seem somewhat mysterious without an appreciation for the methods used to gain it. A brief description of some of the techniques used to purify and detect biochemical compounds may help with understanding how this body of knowledge came about. Thin-layer chromatography is done with paper or a glass plate coated with silica powder or other adsorbent material. A solution containing a mixture of biochemical compounds, ranging from amino acids and nucleic acids to polypeptides, is placed in a spot near one corner. One edge is then placed in contact with a solvent, which carries the compounds along the plate as it is drawn up by capillary action. Compounds with various physicochemical properties move at different speeds. This spreads the compounds out in a line along one edge. That edge is then placed in a different solvent, which moves the compounds across the plate in the other direction. This distributes the individual compounds from the mixture across the two dimensions of the plate (Figure 3.15). The resulting spots can be made visible by chemical treatment or can be removed for further experiments. If the molecules are electrically charged, such as amino acids and polypeptides at the appropriate pH, one of the solvent steps can be replaced with an electrostatic field to move the compounds. This is called electrophoresis. Large molecules and subcellular particles can be separated by sedimentation. This is done in an ultracentrifuge, which creates accelerations up to 400,000g by spinning as fast as 75,000 rpm. Molar masses of large macromolecules can be determined from their sedimentation rate. Particle size is often given in terms of its settling velocity in a centrifuge, measured in Svedberg units. Immunoassay uses antibodies to form a precipitate with specific compounds. Antibodies are special proteins produced by the body to bind with foreign substances so that they can be made harmless. Each antibody is highly specific, binding only to a single substance and binding extremely tightly in what is called a lock-and-key relationship. Molecular biology techniques have enabled the production of large quantities of antibodies of a specific type, called monoclonal antibodies. They are used for research purposes as well as to detect specific hormones in pregnancy tests and tests for prostate cancer. Immunoassay is a highly sensitive and selective detection method. Its use has been extended to organic pollutants and even to heavy metals. The power of the methods described above is often increased by the use of radioisotope labeling. In this technique, radioactive compounds such as 3H, 14C, 32P or 33P, and 35 S are incorporated into substrates, making it easy to detect the products incorporating PROBLEMS 63 1.0 Arg Phe Pro Met Ile Lys Val Leu Trp His Gln 1.0 Ala Thr Asn Tyr Gly Ser 0.3 Glu Asp CySSCy 0.0 0.0 0.3 0.6 1.0 Figure 3.15 Two-dimensional thin-layer chromatograph separation of amino acids. (Based on White et. al., 1973.) them. The radioactivity makes compounds easier to detect in small quantities. For example, exposing plants to carbon dioxide with carbon-14, and then analyzing plant matter periodically for radioactive compounds is a way to elucidate the steps in photosynthesis as the 13C appears in one compound and then another. Radioimmunoassay can detect compounds such as hormones or drugs in blood plasma at quantities as low as several picograms (10
12 g). Other analysis techniques, based on the methods of molecular biology, are discussed in Chapter 6. PROBLEMS 3.1. Write symbol-and-stick diagrams for CO2, NH4, 1,1,1-TCA (1,1,1-trichloroethane), TCE (trichloroethylene), and formic acid (CH2O2). 3.2. Glucose, starch, and glycogen are very similar chemically. How will wastewater containing a high concentration of one or the other affect a biological wastewater treatment plant differently? How would the same wastewater affect a stream differently? Assume that the chemical oxygen demand (COD) of each wastewater is similar. 3.3. A rule of thumb for biological wastewater treatment plant nutrition is that for every 100 mg of oxygen demand there should be 5 mg of nitrogen. (a) Would a wastewater 64 THE SUBSTANCES OF LIFE containing either only glycine or only leucine need a nitrogen supplement? (b) Which of the 20 amino acids is most balanced in this respect? [Hint: Answer by computing the theoretical oxygen demand for each substrate and compare to the mass concentration of nitrogen. The theoretical oxygen demand for CcHhOoNn is c þ (h
3n)/4
o/2 moles of oxygen per mole of substrate. Convert this to grams of oxygen per gram of substrate by the molar mass of O2 (32 g/mol), dividing by the molar mass of the substrate.] Computing oxygen demand is discussed in detail in Section 13.1.3. 3.4. List all the elements that are more electronegative than carbon. 3.5. For each of the acids listed in Table 3.3, compute the fraction that would be undissociated at pH 7.4, the normal pH of human blood.