Water PDF

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8885d_c02_47-74 7/25/03 10:05 AM Page 47 mac76 mac76:385_reb: O – O C 2 H C H chapter WATER 2.1 Weak Interactions in Aqueous Systems 47 and titration curves, and consider how aqueous solu- 2.2 Ionization of Water, Weak Acids, and tions of weak acids or bases and their salts act as buffers Weak Bases 60 against pH changes in biological systems. The water molecule and its ionization products, H
and OH, pro- 2.3 Buffering against pH Changes in Biological foundly influence the structure, self-assembly, and prop- Systems 65 erties of all cellular components, including proteins, 2.4 Water as a Reactant 69 nucleic acids, and lipids. The noncovalent interactions 2.5 The Fitness of the Aqueous Environment responsible for the strength and specificity of “recogni- for Living Organisms 70 tion” among biomolecules are decisively influenced by the solvent properties of water, including its ability to form hydrogen bonds with itself and with solutes. I believe that as the methods of structural chemistry are further applied to physiological problems, it will be found 2.1 Weak Interactions in Aqueous Systems that the significance of the hydrogen bond for physiology Hydrogen bonds between water molecules provide the is greater than that of any other single structural feature. cohesive forces that make water a liquid at room tem- —Linus Pauling, The Nature of the Chemical Bond, 1939 perature and that favor the extreme ordering of mole- cules that is typical of crystalline water (ice). Polar bio- What in water did Bloom, water lover, drawer of water, water molecules dissolve readily in water because they can replace water-water interactions with more energetically carrier returning to the range, admire? Its universality, its favorable water-solute interactions. In contrast, nonpo- democratic quality. lar biomolecules interfere with water-water interactions —James Joyce, Ulysses, 1922 but are unable to form water-solute interactions— consequently, nonpolar molecules are poorly soluble in water. In aqueous solutions, nonpolar molecules tend to ater is the most abundant substance in living sys- W tems, making up 70% or more of the weight of most organisms. The first living organisms doubtless arose in cluster together. Hydrogen bonds and ionic, hydrophobic (Greek, “water-fearing”), and van der Waals interactions are in- an aqueous environment, and the course of evolution dividually weak, but collectively they have a very sig- has been shaped by the properties of the aqueous nificant influence on the three-dimensional structures medium in which life began. of proteins, nucleic acids, polysaccharides, and mem- This chapter begins with descriptions of the physical brane lipids. and chemical properties of water, to which all aspects of cell structure and function are adapted. The attrac- Hydrogen Bonding Gives Water Its Unusual Properties tive forces between water molecules and the slight ten- dency of water to ionize are of crucial importance to the Water has a higher melting point, boiling point, and heat structure and function of biomolecules. We review the of vaporization than most other common solvents (Table topic of ionization in terms of equilibrium constants, pH, 2–1). These unusual properties are a consequence of 47 8885d_c02_47-74 7/25/03 10:05 AM Page 48 mac76 mac76:385_reb: 48 Part I Structure and Catalysis TABLE 2–1 Melting Point, Boiling Point, and Heat of Vaporization of Some Common Solvents Melting point (°C) Boiling point (°C) Heat of vaporization (J/g)* Water 0 100 2,260 Methanol (CH3OH) 98 65 1,100 Ethanol (CH3CH2OH) 117 78 854 Propanol (CH3CH2CH2OH) 127 97 687 Butanol (CH3(CH2)2CH2OH) 90 117 590 Acetone (CH3COCH3) 95 56 523 Hexane (CH3(CH2)4CH3) 98 69 423 Benzene (C6H6) 6 80 394 Butane (CH3(CH2)2CH3) 135 0.5 381 Chloroform (CHCl3) 63 61 247 *The heat energy required to convert 1.0 g of a liquid at its boiling point, at atmospheric pressure, into its gaseous state at the same temperature. It is a direct measure of the energy required to overcome attractive forces between molecules in the liquid phase. attractions between adjacent water molecules that give 
liquid water great internal cohesion. A look at the elec- H tron structure of the H2O molecule reveals the cause of 
these intermolecular attractions. Each hydrogen atom of a water molecule shares an O electron pair with the central oxygen atom. The geom-  etry of the molecule is dictated by the shapes of the 2  H outer electron orbitals of the oxygen atom, which are 

similar to the sp3 bonding orbitals of carbon (see Fig. (a) (b) 1–14). These orbitals describe a rough tetrahedron, with 104.5 a hydrogen atom at each of two corners and unshared electron pairs at the other two corners (Fig. 2–1a). The HOOOH bond angle is 104.5, slightly less than the 109.5 of a perfect tetrahedron because of crowding by the nonbonding orbitals of the oxygen atom. Hydrogen bond The oxygen nucleus attracts electrons more 0.177 nm strongly than does the hydrogen nucleus (a proton); that is, oxygen is more electronegative. The sharing of electrons between H and O is therefore unequal; the Covalent bond 0.0965 nm electrons are more often in the vicinity of the oxygen atom than of the hydrogen. The result of this unequal electron sharing is two electric dipoles in the water mol- ecule, one along each of the HOO bonds; each hydro- (c) gen bears a partial positive charge (

) and the oxygen atom bears a partial negative charge equal to the sum FIGURE 2–1 Structure of the water molecule. The dipolar nature of of the two partial positives (2
). As a result, there is the H2O molecule is shown by (a) ball-and-stick and (b) space-filling an electrostatic attraction between the oxygen atom of models. The dashed lines in (a) represent the nonbonding orbitals. one water molecule and the hydrogen of another (Fig. There is a nearly tetrahedral arrangement of the outer-shell electron 2–1c), called a hydrogen bond. Throughout this book, pairs around the oxygen atom; the two hydrogen atoms have local- we represent hydrogen bonds with three parallel blue ized partial positive charges (
) and the oxygen atom has a partial lines, as in Figure 2–1c. negative charge (2). (c) Two H2O molecules joined by a hydrogen Hydrogen bonds are relatively weak. Those in liq- bond (designated here, and throughout this book, by three blue lines) uid water have a bond dissociation energy (the en- between the oxygen atom of the upper molecule and a hydrogen atom ergy required to break a bond) of about 23 kJ/mol, com- of the lower one. Hydrogen bonds are longer and weaker than cova- pared with 470 kJ/mol for the covalent OOH bond in lent OOH bonds. 8885d_c02_47-74 7/25/03 10:05 AM Page 49 mac76 mac76:385_reb: Chapter 2 Water 49 water or 348 kJ/mol for a covalent COC bond. The hy- drogen bond is about 10% covalent, due to overlaps in the bonding orbitals, and about 90% electrostatic. At room temperature, the thermal energy of an aqueous solution (the kinetic energy of motion of the individual atoms and molecules) is of the same order of magnitude as that required to break hydrogen bonds. When water is heated, the increase in temperature reflects the faster motion of individual water molecules. At any given time, most of the molecules in liquid water are engaged in hy- drogen bonding, but the lifetime of each hydrogen bond is just 1 to 20 picoseconds (1 ps  1012 s); upon break- age of one hydrogen bond, another hydrogen bond forms, with the same partner or a new one, within 0.1 ps. The apt phrase “flickering clusters” has been applied to the short-lived groups of water molecules interlinked by hydrogen bonds in liquid water. The sum of all the hy- drogen bonds between H2O molecules confers great in- ternal cohesion on liquid water. Extended networks of hydrogen-bonded water molecules also form bridges be- tween solutes (proteins and nucleic acids, for example) that allow the larger molecules to interact with each FIGURE 2–2 Hydrogen bonding in ice. In ice, each water molecule other over distances of several nanometers without forms the maximum of four hydrogen bonds, creating a regular crys- physically touching. tal lattice. By contrast, in liquid water at room temperature and at- The nearly tetrahedral arrangement of the orbitals mospheric pressure, each water molecule hydrogen-bonds with an av- about the oxygen atom (Fig. 2–1a) allows each water erage of 3.4 other water molecules. This crystal lattice of ice makes it molecule to form hydrogen bonds with as many as four less dense than liquid water, and thus ice floats on liquid water. neighboring water molecules. In liquid water at room temperature and atmospheric pressure, however, water molecules are disorganized and in continuous motion, and breaking bonds, and S the change in randomness. so that each molecule forms hydrogen bonds with an av- Because H is positive for melting and evaporation, it erage of only 3.4 other molecules. In ice, on the other is clearly the increase in entropy (S) that makes G hand, each water molecule is fixed in space and forms negative and drives these transformations. hydrogen bonds with a full complement of four other water molecules to yield a regular lattice structure (Fig. Water Forms Hydrogen Bonds with Polar Solutes 2–2). Breaking a sufficient proportion of hydrogen bonds to destabilize the crystal lattice of ice requires Hydrogen bonds are not unique to water. They readily much thermal energy, which accounts for the relatively form between an electronegative atom (the hydrogen high melting point of water (Table 2–1). When ice melts acceptor, usually oxygen or nitrogen with a lone pair of or water evaporates, heat is taken up by the system: electrons) and a hydrogen atom covalently bonded to another electronegative atom (the hydrogen donor) in H2O(solid) 88n H2O(liquid) H 
5.9 kJ/mol the same or another molecule (Fig. 2–3). Hydrogen atoms covalently bonded to carbon atoms do not par- H2O(liquid) 88n H2O(gas) H 
44.0 kJ/mol ticipate in hydrogen bonding, because carbon is only During melting or evaporation, the entropy of the aqueous system increases as more highly ordered arrays of water molecules relax into the less orderly hydrogen- G D G D C C P P bonded arrays in liquid water or the wholly disordered Hydrogen J D D D D D J D acceptor O N O O O N gaseous state. At room temperature, both the melting of ice and the evaporation of water occur spontaneously; Hydrogen H H H H H H O O O O O O O O O O O O the tendency of the water molecules to associate through donor O O O N N N hydrogen bonds is outweighed by the energetic push toward randomness. Recall that the free-energy change (G) must have a negative value for a process to occur FIGURE 2–3 Common hydrogen bonds in biological systems. The spontaneously: G  H  T S, where G represents hydrogen acceptor is usually oxygen or nitrogen; the hydrogen donor the driving force, H the enthalpy change from making is another electronegative atom. 8885d_c02_47-74 7/25/03 10:05 AM Page 50 mac76 mac76:385_reb: 50 Part I Structure and Catalysis slightly more electronegative than hydrogen and thus R A R the COH bond is only very weakly polar. The distinc- O A tion explains why butanol (CH3(CH2)2CH2OH) has a rel- A O H Strong A atively high boiling point of 117 C, whereas butane H Weaker hydrogen bond (CH3(CH2)2CH3) has a boiling point of only 0.5 C. Bu- G KO G KO hydrogen bond OP OP tanol has a polar hydroxyl group and thus can form in- D D termolecular hydrogen bonds. Uncharged but polar bio- molecules such as sugars dissolve readily in water FIGURE 2–5 Directionality of the hydrogen bond. The attraction be- because of the stabilizing effect of hydrogen bonds be- tween the partial electric charges (see Fig. 2–1) is greatest when the tween the hydroxyl groups or carbonyl oxygen of the three atoms involved (in this case O, H, and O) lie in a straight line. sugar and the polar water molecules. Alcohols, alde- When the hydrogen-bonded moieties are structurally constrained (as hydes, ketones, and compounds containing NOH bonds when they are parts of a single protein molecule, for example), this all form hydrogen bonds with water molecules (Fig. 2–4) ideal geometry may not be possible and the resulting hydrogen bond and tend to be soluble in water. is weaker. Hydrogen bonds are strongest when the bonded molecules are oriented to maximize electrostatic inter- action, which occurs when the hydrogen atom and the ing two hydrogen-bonded molecules or groups in a spe- two atoms that share it are in a straight line—that is, cific geometric arrangement. As we shall see later, this when the acceptor atom is in line with the covalent bond property of hydrogen bonds confers very precise three- between the donor atom and H (Fig. 2–5). Hydrogen dimensional structures on protein and nucleic acid bonds are thus highly directional and capable of hold- molecules, which have many intramolecular hydrogen bonds. Water Interacts Electrostatically Between the Between the Between peptide with Charged Solutes hydroxyl group carbonyl group groups in of an alcohol of a ketone polypeptides Water is a polar solvent. It readily dissolves most bio- and water and water molecules, which are generally charged or polar com- H R pounds (Table 2–2); compounds that dissolve easily in R R1 R2 A A G G D NH EC water are hydrophilic (Greek, “water-loving”). In con- O C trast, nonpolar solvents such as chloroform and benzene A B C H B are poor solvents for polar biomolecules but easily dis- H O O solve those that are hydrophobic—nonpolar molecules E OH H A H such as lipids and waxes. H H A OH Water dissolves salts such as NaCl by hydrating and HENH H C C stabilizing the Na
and Cl ions, weakening the elec- A B R O trostatic interactions between them and thus counter- acting their tendency to associate in a crystalline lattice Between (Fig. 2–6). The same factors apply to charged biomole- complementary cules, compounds with functional groups such as ion- bases of DNA ized carboxylic acids (OCOO), protonated amines H (ONH
3 ), and phosphate esters or anhydrides. Water A readily dissolves such compounds by replacing solute- R H E N ECH3 C solute hydrogen bonds with solute-water hydrogen N C A A Thymine bonds, thus screening the electrostatic interactions be- KCH EC N tween solute molecules. O N O A Water is especially effective in screening the elec- H H trostatic interactions between dissolved ions because it A H has a high dielectric constant, a physical property re- H E NN ENH C C flecting the number of dipoles in a solvent. The strength, B A Adenine or force (F), of ionic interactions in a solution depends N C H K H upon the magnitude of the charges (Q), the distance C N i l between the charged groups (r), and the dielectric con- NOCH E stant () of the solvent in which the interactions occur: R Q1Q2 F  FIGURE 2–4 Some biologically important hydrogen bonds. r2 8885d_c02_051 7/25/03 11:52 AM Page 51 mac76 mac76:385_reb: Chapter 2 Water 51 TABLE 2–2 Some Examples of Polar, Nonpolar, and Amphipathic Biomolecules (Shown as Ionic Forms at pH 7) Polar Nonpolar O Glucose CH2OH Typical wax CH3(CH2)7 CH CH (CH2)6 CH2 C O H OH O H CH3 (CH2)7 CH CH (CH2)7 CH2 OH H H HO H OH Amphipathic Phenylalanine GNH3 NH COO Glycine 3 CH2 CH2 CH COOJ Aspartate NH 3 OOC CH2 CH COO Phosphatidylcholine O Lactate CH3 CH COO CH3(CH2)15CH2 C O CH2 OH CH3(CH2)15CH2 C O CH O GN(CH3)3 O CH2 O P O CH2 CH2 Glycerol OH OJ HOCH2 CH CH2OH Polar groups Nonpolar groups For water at 25
C,
(which is dimensionless) is 78.5, and for the very nonpolar solvent benzene,
is 4.6. Thus, Entropy Increases as Crystalline Substances Dissolve ionic interactions are much stronger in less polar envi- As a salt such as NaCl dissolves, the Na and Cl ions ronments. The dependence on r 2 is such that ionic at- leaving the crystal lattice acquire far greater freedom of tractions or repulsions operate only over short dis- motion (Fig. 2–6). The resulting increase in entropy tances—in the range of 10 to 40 nm (depending on the (randomness) of the system is largely responsible for electrolyte concentration) when the solvent is water. the ease of dissolving salts such as NaCl in water. In H2O + Hydrated + Na+ ion Na+ – – – + Note the orientation + – of the water molecules + – – – + + Cl– – – – Hydrated Cl– ion – – + + – – – – – FIGURE 2–6 Water as solvent. Water dissolves many crystalline salts charges are partially neutralized, and the electrostatic attractions nec- by hydrating their component ions. The NaCl crystal lattice is disrupted essary for lattice formation are weakened. as water molecules cluster about the Cl and Na ions. The ionic 8885d_c02_47-74 7/25/03 10:05 AM Page 52 mac76 mac76:385_reb: 52 Part I Structure and Catalysis thermodynamic terms, formation of the solution occurs hydrophobic—they are unable to undergo energetically with a favorable free-energy change: G  H  T S, favorable interactions with water molecules, and they where H has a small positive value and T S a large interfere with the hydrogen bonding among water mol- positive value; thus G is negative. ecules. All molecules or ions in aqueous solution inter- fere with the hydrogen bonding of some water mole- cules in their immediate vicinity, but polar or charged Nonpolar Gases Are Poorly Soluble in Water solutes (such as NaCl) compensate for lost water-water The molecules of the biologically important gases CO2, hydrogen bonds by forming new solute-water interac- O2, and N2 are nonpolar. In O2 and N2, electrons are tions. The net change in enthalpy (H) for dissolving shared equally by both atoms. In CO2, each CUO bond these solutes is generally small. Hydrophobic solutes, is polar, but the two dipoles are oppositely directed and however, offer no such compensation, and their addi- cancel each other (Table 2–3). The movement of mole- tion to water may therefore result in a small gain of en- cules from the disordered gas phase into aqueous solu- thalpy; the breaking of hydrogen bonds between water tion constrains their motion and the motion of water molecules takes up energy from the system. Further- molecules and therefore represents a decrease in en- more, dissolving hydrophobic compounds in water pro- tropy. The nonpolar nature of these gases and the de- duces a measurable decrease in entropy. Water mole- crease in entropy when they enter solution combine to cules in the immediate vicinity of a nonpolar solute are make them very poorly soluble in water (Table 2–3). constrained in their possible orientations as they form Some organisms have water-soluble carrier proteins a highly ordered cagelike shell around each solute mol- (hemoglobin and myoglobin, for example) that facilitate ecule. These water molecules are not as highly oriented the transport of O2. Carbon dioxide forms carbonic acid as those in clathrates, crystalline compounds of non- (H2CO3) in aqueous solution and is transported as the polar solutes and water, but the effect is the same in HCO 3 (bicarbonate) ion, either free—bicarbonate is both cases: the ordering of water molecules reduces en- very soluble in water (~100 g/L at 25 C)—or bound to tropy. The number of ordered water molecules, and hemoglobin. Two other gases, NH3 and H2S, also have therefore the magnitude of the entropy decrease, is pro- biological roles in some organisms; these gases are po- portional to the surface area of the hydrophobic solute lar and dissolve readily in water. enclosed within the cage of water molecules. The free- energy change for dissolving a nonpolar solute in water Nonpolar Compounds Force Energetically Unfavorable is thus unfavorable: G  H  T S, where H has a positive value, S has a negative value, and G is Changes in the Structure of Water positive. When water is mixed with benzene or hexane, two Amphipathic compounds contain regions that are phases form; neither liquid is soluble in the other. Non- polar (or charged) and regions that are nonpolar (Table polar compounds such as benzene and hexane are 2–2). When an amphipathic compound is mixed with TABLE 2–3 Solubilities of Some Gases in Water Solubility Gas Structure* Polarity in water (g/L)† Nitrogen NmN Nonpolar 0.018 (40 °C) Oxygen OPO Nonpolar 0.035 (50 °C) Carbon dioxide   Nonpolar 0.97 (45 °C) OPCP O Ammonia H H A H Polar 900 (10 °C) G D N  Hydrogen sulfide H H Polar 1,860 (40 °C) G D S  *The arrows represent electric dipoles; there is a partial negative charge (
) at the head of the arrow, a partial positive charge (

; not shown here) at the tail. † Note that polar molecules dissolve far better even at low temperatures than do nonpolar molecules at relatively high temperatures. 8885d_c02_47-74 7/25/03 10:05 AM Page 53 mac76 mac76:385_reb: Chapter 2 Water 53 Hydrophilic O – “head group” O H C Dispersion of H O lipids in H2O H C H Each lipid molecule forces surrounding H2O molecules to become highly ordered. Hydrophobic alkyl group “Flickering clusters” of H2O molecules in bulk phase Highly ordered H2O molecules form “cages” around the hydrophobic alkyl chains (a) Clusters of lipid molecules FIGURE 2–7 Amphipathic compounds in aqueous solution. (a) Long- Only lipid portions chain fatty acids have very hydrophobic alkyl chains, each of which at the edge of the cluster force the is surrounded by a layer of highly ordered water molecules. (b) By ordering of water. clustering together in micelles, the fatty acid molecules expose the Fewer H2O molecules smallest possible hydrophobic surface area to the water, and fewer are ordered, and entropy is increased. water molecules are required in the shell of ordered water. The energy gained by freeing immobilized water molecules stabilizes the micelle. water, the polar, hydrophilic region interacts favorably Micelles with the solvent and tends to dissolve, but the nonpo- All hydrophobic groups are lar, hydrophobic region tends to avoid contact with the sequestered from water (Fig. 2–7a). The nonpolar regions of the mole- water; ordered cules cluster together to present the smallest hy- shell of H2O molecules is drophobic area to the aqueous solvent, and the polar re- minimized, and gions are arranged to maximize their interaction with entropy is further the solvent (Fig. 2–7b). These stable structures of am- increased. phipathic compounds in water, called micelles, may contain hundreds or thousands of molecules. The forces (b) that hold the nonpolar regions of the molecules together are called hydrophobic interactions. The strength of hydrophobic interactions is not due to any intrinsic at- traction between nonpolar moieties. Rather, it results from the system’s achieving greatest thermodynamic polar regions. Hydrophobic interactions among lipids, stability by minimizing the number of ordered water and between lipids and proteins, are the most impor- molecules required to surround hydrophobic portions of tant determinants of structure in biological membranes. the solute molecules. Hydrophobic interactions between nonpolar amino Many biomolecules are amphipathic; proteins, pig- acids also stabilize the three-dimensional structures of ments, certain vitamins, and the sterols and phospho- proteins. lipids of membranes all have polar and nonpolar surface Hydrogen bonding between water and polar solutes regions. Structures composed of these molecules are also causes some ordering of water molecules, but the stabilized by hydrophobic interactions among the non- effect is less significant than with nonpolar solutes. Part 8885d_c02_47-74 7/25/03 10:05 AM Page 54 mac76 mac76:385_reb: 54 Part I Structure and Catalysis Ordered water other. Random variations in the positions of the electrons interacting with around one nucleus may create a transient electric di- substrate and enzyme pole, which induces a transient, opposite electric dipole in the nearby atom. The two dipoles weakly attract each other, bringing the two nuclei closer. These weak at- tractions are called van der Waals interactions. As Substrate the two nuclei draw closer together, their electron clouds begin to repel each other. At the point where the van der Waals attraction exactly balances this repulsive force, the nuclei are said to be in van der Waals contact. Enzyme Each atom has a characteristic van der Waals radius, a measure of how close that atom will allow another to approach (Table 2–4). In the “space-filling” molecular models shown throughout this book, the atoms are de- picted in sizes proportional to their van der Waals radii. Weak Interactions Are Crucial to Macromolecular Structure and Function The noncovalent interactions we have described (hy- drogen bonds and ionic, hydrophobic, and van der Waals interactions) (Table 2–5) are much weaker than cova- Disordered water lent bonds. An input of about 350 kJ of energy is re- displaced by quired to break a mole of (6 1023) COC single bonds, enzyme-substrate interaction and about 410 kJ to break a mole of COH bonds, but as little as 4 kJ is sufficient to disrupt a mole of typical van der Waals interactions. Hydrophobic interactions are also much weaker than covalent bonds, although they are substantially strengthened by a highly polar sol- vent (a concentrated salt solution, for example). Ionic interactions and hydrogen bonds are variable in strength, depending on the polarity of the solvent and Enzyme-substrate interaction stabilized by hydrogen-bonding, ionic, and hydrophobic interactions TABLE 2–4 van der Waals Radii and Covalent FIGURE 2–8 Release of ordered water favors formation of an (Single-Bond) Radii of Some Elements enzyme-substrate complex. While separate, both enzyme and sub- van der Waals Covalent radius for strate force neighboring water molecules into an ordered shell. Bind- Element radius (nm) single bond (nm) ing of substrate to enzyme releases some of the ordered water, and the resulting increase in entropy provides a thermodynamic push to- H 0.11 0.030 ward formation of the enzyme-substrate complex. O 0.15 0.066 N 0.15 0.070 C 0.17 0.077 S 0.18 0.104 P 0.19 0.110 of the driving force for binding of a polar substrate (re- I 0.21 0.133 actant) to the complementary polar surface of an en- zyme is the entropy increase as the enzyme displaces Sources: For van der Waals radii, Chauvin, R. (1992) Explicit periodic trend of van der ordered water from the substrate (Fig. 2–8). Waals radii. J. Phys. Chem. 96, 9194–9197. For covalent radii, Pauling, L. (1960) Nature of the Chemical Bond, 3rd edn, Cornell University Press, Ithaca, NY. van der Waals Interactions Are Weak Note: van der Waals radii describe the space-filling dimensions of atoms. When two atoms are joined covalently, the atomic radii at the point of bonding are less than the van der Interatomic Attractions Waals radii, because the joined atoms are pulled together by the shared electron pair. The distance between nuclei in a van der Waals interaction or a covalent bond is about equal When two uncharged atoms are brought very close to- to the sum of the van der Waals or covalent radii, respectively, for the two atoms. Thus the gether, their surrounding electron clouds influence each length of a carbon-carbon single bond is about 0.077 nm
0.077 nm  0.154 nm. 8885d_c02_47-74 7/25/03 10:05 AM Page 55 mac76 mac76:385_reb: Chapter 2 Water 55 through multiple weak interactions requires all these in- TABLE 2–5 Four Types of Noncovalent (“Weak”) teractions to be disrupted at the same time. Because Interactions among Biomolecules in Aqueous Solvent the interactions fluctuate randomly, such simultaneous disruptions are very unlikely. The molecular stability be- Hydrogen bonds G stowed by 5 or 20 weak interactions is therefore much Between neutral groups CPO HO OO D greater than would be expected intuitively from a sim- ple summation of small binding energies. G G Macromolecules such as proteins, DNA, and RNA Between peptide bonds CPO HON D D contain so many sites of potential hydrogen bonding or ionic, van der Waals, or hydrophobic interactions that Ionic interactions O the cumulative effect of the many small binding forces B can be enormous. For macromolecules, the most stable Attraction O
NH3 O O CO (that is, the native) structure is usually that in which weak-bonding possibilities are maximized. The folding Repulsion O
NH3 H3N
O of a single polypeptide or polynucleotide chain into its three-dimensional shape is determined by this princi- ple. The binding of an antigen to a specific antibody de- water pends on the cumulative effects of many weak interac- CH3 CH3 tions. As noted earlier, the energy released when an G D CH enzyme binds noncovalently to its substrate is the main Hydrophobic interactions A CH2 source of the enzyme’s catalytic power. The binding of A a hormone or a neurotransmitter to its cellular recep- CH2 tor protein is the result of weak interactions. One con- A sequence of the large size of enzymes and receptors is that their extensive surfaces provide many opportuni- van der Waals interactions Any two atoms in ties for weak interactions. At the molecular level, the close proximity complementarity between interacting biomolecules re- flects the complementarity and weak interactions be- tween polar, charged, and hydrophobic groups on the surfaces of the molecules. When the structure of a protein such as hemoglobin the alignment of the hydrogen-bonded atoms, but they (Fig. 2–9) is determined by x-ray crystallography (see are always significantly weaker than covalent bonds. In aqueous solvent at 25 C, the available thermal energy can be of the same order of magnitude as the strength of these weak interactions, and the interaction between solute and solvent (water) molecules is nearly as favor- able as solute-solute interactions. Consequently, hydro- gen bonds and ionic, hydrophobic, and van der Waals interactions are continually formed and broken. Although these four types of interactions are indi- vidually weak relative to covalent bonds, the cumulative effect of many such interactions can be very significant. For example, the noncovalent binding of an enzyme to its substrate may involve several hydrogen bonds and one or more ionic interactions, as well as hydrophobic and van der Waals interactions. The formation of each of these weak bonds contributes to a net decrease in the free energy of the system. We can calculate the sta- (a) (b) bility of a noncovalent interaction, such as that of a small FIGURE 2–9 Water binding in hemoglobin. The crystal structure of molecule hydrogen-bonded to its macromolecular part- hemoglobin, shown (a) with bound water molecules (red spheres) and ner, from the binding energy. Stability, as measured by (b) without the water molecules. These water molecules are so firmly the equilibrium constant (see below) of the binding re- bound to the protein that they affect the x-ray diffraction pattern as action, varies exponentially with binding energy. The though they were fixed parts of the crystal. The gray structures with dissociation of two biomolecules (such as an enzyme red and orange atoms are the four hemes of hemoglobin, discussed and its bound substrate) associated noncovalently in detail in Chapter 5. 8885d_c02_47-74 7/25/03 10:05 AM Page 56 mac76 mac76:385_reb: 56 Part I Structure and Catalysis Box 4–4, p. XX), water molecules are often found to be Solutes Affect the Colligative Properties bound so tightly as to be part of the crystal structure; of Aqueous Solutions the same is true for water in crystals of RNA or DNA. These bound water molecules, which can also be de- Solutes of all kinds alter certain physical properties of tected in aqueous solutions by nuclear magnetic reso- the solvent, water: its vapor pressure, boiling point, nance, have distinctly different properties from those of melting point (freezing point), and osmotic pressure. the “bulk” water of the solvent. They are, for example, These are called colligative (“tied together”) proper- not osmotically active (see below). For many proteins, ties, because the effect of solutes on all four properties tightly bound water molecules are essential to their func- has the same basis: the concentration of water is lower tion. In a reaction central to the process of photosyn- in solutions than in pure water. The effect of solute con- thesis, for example, light drives protons across a biolog- centration on the colligative properties of water is in- ical membrane as electrons flow through a series of dependent of the chemical properties of the solute; it electron-carrying proteins (see Fig. 19–XX). One of these depends only on the number of solute particles (mole- proteins, cytochrome f, has a chain of five bound water cules, ions) in a given amount of water. A compound molecules (Fig. 2–10) that may provide a path for pro- such as NaCl, which dissociates in solution, has twice tons to move through the membrane by a process known the effect on osmotic pressure, for example, as does an as “proton hopping” (described below). Another such equal number of moles of a nondissociating solute such light-driven proton pump, bacteriorhodopsin, almost cer- as glucose. tainly uses a chain of precisely oriented bound water Solutes alter the colligative properties of aqueous molecules in the transmembrane movement of protons solutions by lowering the effective concentration of wa- (see Fig. 19–XX). ter. For example, when a significant fraction of the mol- ecules at the surface of an aqueous solution are not wa- ter but solute, the tendency of water molecules to escape into the vapor phase—that is, the vapor pres- sure—is lowered (Fig. 2–11). Similarly, the tendency of Val60 Pro231 Gln59 water molecules to move from the aqueous phase to the H H O – O Heme surface of a forming ice crystal is reduced when some N H propionate of the molecules that collide with the crystal are solute, water O H not water. In that case, the solution will freeze more O H slowly than pure water and at a lower temperature. For N O Asn232 Asn168 a 1.00 molal aqueous solution (1.00 mol of solute per H HN 1,000 g of water) of an ideal, nonvolatile, and nondis- O sociating solute at 101 kPa (1 atm) of pressure, the H Arg156 O freezing point is 1.86 C lower and the boiling point is Asn153 H 0.543 C higher than for pure water. For a 0.100 molal HN O NH2 solution of the same solute, the changes are one-tenth N H H Gln158 as large. O N Water molecules tend to move from a region of N higher water concentration to one of lower water con- Ala27 Fe centration. When two different aqueous solutions are separated by a semipermeable membrane (one that al- H N H lows the passage of water but not solute molecules), wa- HO C C ter molecules diffusing from the region of higher water H concentration to that of lower water concentration pro- O duce osmotic pressure (Fig. 2–12). This pressure, , measured as the force necessary to resist water move- FIGURE 2–10 Water chain in cytochrome f. Water is bound in a pro- ment (Fig. 2–12c), is approximated by the van’t Hoff ton channel of the membrane protein cytochrome f, which is part of equation: the energy-trapping machinery of photosynthesis in chloroplasts (see Fig. 19–XX). Five water molecules are hydrogen-bonded to each other  icRT and to functional groups of the protein, which include the side chains of valine, proline, arginine, alanine, two asparagine, and two gluta- in which R is the gas constant and T is the absolute tem- mine residues. The protein has a bound heme (see Fig. 5–1), its iron perature. The term ic is the osmolarity of the solution, ion facilitating electron flow during photosynthesis. Electron flow is the product of the solute’s molar concentration c and coupled to the movement of protons across the membrane, which the van’t Hoff factor i, which is a measure of the extent probably involves “electron hopping” (see Fig. 2–14) through this to which the solute dissociates into two or more ionic chain of bound water molecules. species. In dilute NaCl solutions, the solute completely 8885d_c02_47-74 7/25/03 10:05 AM Page 57 mac76 mac76:385_reb: Chapter 2 Water 57 = H2O osmolarity than the cytosol, the cell shrinks as water = Solute flows out. In a hypotonic solution, with lower osmo- larity than the cytosol, the cell swells as water enters. Forming ice crystal In their natural environments, cells generally contain higher concentrations of biomolecules and ions than their surroundings, so osmotic pressure tends to drive water into cells. If not somehow counterbalanced, this inward movement of water would distend the plasma membrane and eventually cause bursting of the cell (osmotic lysis). Several mechanisms have evolved to prevent this catastrophe. In bacteria and plants, the plasma mem- brane is surrounded by a nonexpandable cell wall of suf- ficient rigidity and strength to resist osmotic pressure and prevent osmotic lysis. Certain freshwater protists (a) (b) that live in a highly hypotonic medium have an organelle In pure water, every In this solution, the (contractile vacuole) that pumps water out of the cell. molecule at the surface is effective concentration of In multicellular animals, blood plasma and interstitial H2O, and all contribute H2O is reduced; only 3 of to the vapor pressure. every 4 molecules at the fluid (the extracellular fluid of tissues) are maintained Every molecule in the bulk surface and in the bulk at an osmolarity close to that of the cytosol. The high solution is H2O, and can phase are H2O. The vapor concentration of albumin and other proteins in blood contribute to formation of pressure of water and the ice crystals. tendency of liquid water to plasma contributes to its osmolarity. Cells also actively enter a crystal are reduced pump out ions such as Na
into the interstitial fluid to proportionately. stay in osmotic balance with their surroundings. FIGURE 2–11 Solutes alter the colligative properties of aqueous so- lutions. (a) At 101 kPa (1 atm) pressure, pure water boils at 100 C and freezes at 0 C. (b) The presence of solute molecules reduces the Pure Nonpermeant probability of a water molecule leaving the solution and entering the water solute dissolved Piston gas phase, thereby reducing the vapor pressure of the solution and in- in water creasing the boiling point. Similarly, the probability of a water mole- cule colliding with and joining a forming ice crystal is reduced when some of the molecules colliding with the crystal are solute, not wa- h ter, molecules. The effect is depression of the freezing point. dissociates into Na
and Cl, doubling the number of solute particles, and thus i  2. For nonionizing solutes, (a) (b) (c) i is always 1. For solutions of several (n) solutes, is the sum of the contributions of each species: Semipermeable membrane  RT(i1c1
i2c2
…
incn) FIGURE 2–12 Osmosis and the measurement of osmotic pressure. Osmosis, water movement across a semipermeable (a) The initial state. The tube contains an aqueous solution, the beaker membrane driven by differences in osmotic pressure, is contains pure water, and the semipermeable membrane allows the an important factor in the life of most cells. Plasma passage of water but not solute. Water flows from the beaker into the membranes are more permeable to water than to most tube to equalize its concentration across the membrane. (b) The final other small molecules, ions, and macromolecules. This state. Water has moved into the solution of the nonpermeant com- permeability is due partly to simple diffusion of water pound, diluting it and raising the column of water within the tube. At through the lipid bilayer and partly to protein channels equilibrium, the force of gravity operating on the solution in the tube (aquaporins; see Fig. 11–XX) in the membrane that se- exactly balances the tendency of water to move into the tube, where lectively permit the passage of water. Solutions of equal its concentration is lower. (c) Osmotic pressure ( ) is measured as the osmolarity are said to be isotonic. Surrounded by an force that must be applied to return the solution in the tube to the isotonic solution, a cell neither gains nor loses water level of that in the beaker. This force is proportional to the height, h, (Fig. 2–13). In a hypertonic solution, one with higher of the column in (b). 8885d_c02_47-74 7/25/03 10:05 AM Page 58 mac76 mac76:385_reb: 58 Part I Structure and Catalysis Extracellular parts seen in touch-sensitive plants such as the Venus solutes flytrap and mimosa (Box 2–1). Intracellular solutes Osmosis also has consequences for laboratory pro- tocols. Mitochondria, chloroplasts, and lysosomes, for ex- (a) Cell in isotonic ample, are bounded by semipermeable membranes. In solution; no net water isolating these organelles from broken cells, biochemists movement. must perform the fractionations in isotonic solutions (see Fig. 1–8). Buffers used in cellular fractionations commonly contain sufficient concentrations (about 0.2 M) of sucrose or some other inert solute to protect the organelles from osmotic lysis. SUMMARY 2.1 Weak Interactions in Aqueous Systems The very different electronegativities of H and O make water a highly polar molecule, capable of forming hydrogen bonds with itself and with solutes. Hydrogen bonds are fleeting, primarily (b) Cell in hypertonic (c) Cell in hypotonic electrostatic, and weaker than covalent bonds. solution; water moves out solution; water moves in, Water is a good solvent for polar (hydrophilic) and cell shrinks. creating outward pressure; solutes, with which it forms hydrogen bonds, cell swells, may eventually burst. and for charged solutes, with which it interacts electrostatically. Nonpolar (hydrophobic) compounds dissolve FIGURE 2–13 Effect of extracellular osmolarity on water movement poorly in water; they cannot hydrogen-bond across a plasma membrane. When a cell in osmotic balance with its with the solvent, and their presence forces an surrounding medium (that is, in an isotonic medium) (a) is transferred energetically unfavorable ordering of water into a hypertonic solution (b) or hypotonic solution (c), water moves molecules at their hydrophobic surfaces. To across the plasma membrane in the direction that tends to equalize minimize the surface exposed to water, nonpolar osmolarity outside and inside the cell. compounds such as lipids form aggregates (micelles) in which the hydrophobic moieties are sequestered in the interior, associating through hydrophobic interactions, and only the more polar moieties interact with water. Because the effect of solutes on osmolarity depends on the number of dissolved particles, not their mass, Numerous weak, noncovalent interactions deci- macromolecules (proteins, nucleic acids, polysaccha- sively influence the folding of macromolecules rides) have far less effect on the osmolarity of a solu- such as proteins and nucleic acids. The most tion than would an equal mass of their monomeric com- stable macromolecular conformations are those ponents. For example, a gram of a polysaccharide in which hydrogen bonding is maximized within composed of 1,000 glucose units has the same effect on the molecule and between the molecule and osmolarity as a milligram of glucose. One effect of stor- the solvent, and in which hydrophobic moieties ing fuel as polysaccharides (starch or glycogen) rather cluster in the interior of the molecule away than as glucose or other simple sugars is prevention of from the aqueous solvent. an enormous increase in osmotic pressure within the The physical properties of aqueous solutions storage cell. are strongly influenced by the concentrations Plants use osmotic pressure to achieve mechanical of solutes. When two aqueous compartments rigidity. The very high solute concentration in the plant are separated by a semipermeable membrane cell vacuole draws water into the cell (Fig. 2–13). The (such as the plasma membrane separating a resulting osmotic pressure against the cell wall (turgor cell from its surroundings), water moves across pressure) stiffens the cell, the tissue, and the plant body. that membrane to equalize the osmolarity in When the lettuce in your salad wilts, it is because loss the two compartments. This tendency for water of water has reduced turgor pressure. Sudden alter- to move across a semipermeable membrane is ations in turgor pressure produce the movement of plant the osmotic pressure. 8885d_c02_47-74 7/25/03 10:05 AM Page 59 mac76 mac76:385_reb: Chapter 2 Water 59 BOX 2–1 THE WORLD OF BIOCHEMISTRY Touch Response in Plants: An Osmotic Event cells and the resulting efflux, by osmosis, of water. Di- The highly specialized leaves of the Venus flytrap gestive glands in the leaf’s surface release enzymes (Dionaea muscipula) rapidly fold together in re- that extract nutrients from the insect. sponse to a light touch by an unsuspecting insect, en- The sensitive plant (Mimosa pudica) also un- trapping the insect for later digestion. Attracted by dergoes a remarkable change in leaf shape triggered nectar on the leaf surface, the insect touches three by mechanical touch (Fig. 2). A light touch or vibra- mechanically sensitive hairs, triggering the traplike tion produces a sudden drooping of the leaves, the re- closing of the leaf (Fig. 1). This leaf movement is pro- sult of a dramatic reduction in turgor pressure in cells duced by sudden (within 0.5 s) changes of turgor pres- at the base of each leaflet and leaf. As in the Venus sure in mesophyll cells (the inner cells of the leaf), flytrap, the drop in turgor pressure results from K
probably achieved by the release of K
ions from the release followed by the efflux of water. FIGURE 1 Touch response in the Venus flytrap. A fly approaching an open leaf (a) is trapped for digestion (a) (b) by the plant (b). FIGURE 2 The feathery leaflets of the sensitive plant (a) close and drop (b) to protect the plant from (a) (b) structural damage by wind. 8885d_c02_47-74 7/25/03 10:05 AM Page 60 mac76 mac76:385_reb: 60 Part I Structure and Catalysis 2.2 Ionization of Water, Weak Acids, Hydronium ion gives up a proton H H and Weak Bases O+ Proton hop Although many of the solvent properties of water can H be explained in terms of the uncharged H2O molecule, O H H the small degree of ionization of water to hydrogen ions H O H (H
) and hydroxide ions (OH) must also be taken into H O H account. Like all reversible reactions, the ionization of water can be described by an equilibrium constant. H O When weak acids are dissolved in water, they contribute H H
by ionizing; weak bases consume H
by becoming protonated. These processes are also governed by equi- O H librium constants. The total hydrogen ion concentration H H from all sources is experimentally measurable and is ex- O pressed as the pH of the solution. To predict the state of ionization of solutes in water, we must take into ac- H count the relevant equilibrium constants for each ion- O H ization reaction. We therefore turn now to a brief dis- cussion of the ionization of water and of weak acids and H bases dissolved in water. H O Pure Water Is Slightly Ionized H O H Water molecules have a slight tendency to undergo re- H versible ionization to yield a hydrogen ion (a proton) Water accepts proton and and a hydroxide ion, giving the equilibrium becomes a hydronium ion z H

OH H2O y (2–1) FIGURE 2–14 Proton hopping. Short “hops” of protons between a se- Although we commonly show the dissociation product ries of hydrogen-bonded water molecules effect an extremely rapid of water as H
, free protons do not exist in solution; hy- net movement of a proton over a long distance. As a hydronium ion drogen ions formed in water are immediately hydrated (upper left) gives up a proton, a water molecule some distance away to hydronium ions (H3O
). Hydrogen bonding be- (lower right) acquires one, becoming a hydronium ion. Proton hop- tween water molecules makes the hydration of dissoci- ping is much faster than true diffusion and explains the remarkably ating protons virtually instantaneous: high ionic mobility of H
ions compared with other monovalent cations such as Na
or K
. H O H O H O
H
OH H H H The ionization of water can be measured by its elec- trical conductivity; pure water carries electrical current expressing the extent of ionization of water in quanti- as H
migrates toward the cathode and OH toward the tative terms. A brief review of some properties of re- anode. The movement of hydronium and hydroxide ions versible chemical reactions shows how this can be done. in the electric field is anomalously fast compared with The position of equilibrium of any chemical reac- that of other ions such as Na
, K
, and Cl. This high tion is given by its equilibrium constant, Keq (some- ionic mobility results from the kind of “proton hopping” times expressed simply as K ). For the generalized shown in Figure 2–14. No individual proton moves very reaction far through the bulk solution, but a series of proton hops A
B y z C
D (2–2) between hydrogen-bonded water molecules causes the net movement of a proton over a long distance in a re- an equilibrium constant can be defined in terms of the markably short time. As a result of the high ionic mo- concentrations of reactants (A and B) and products (C bility of H
(and of OH, which also moves rapidly by and D) at equilibrium: proton hopping, but in the opposite direction), acid-base [C][D] reactions in aqueous solutions are generally exception- Keq   [A][B] ally fast. As noted above, proton hopping very likely also plays a role in biological proton-transfer reactions (Fig. Strictly speaking, the concentration terms should be 2–10; see also Fig. 19–XX). the activities, or effective concentrations in nonideal Because reversible ionization is crucial to the role solutions, of each species. Except in very accurate work, of water in cellular function, we must have a means of however, the equilibrium constant may be approxi- 8885d_c02_47-74 7/25/03 10:05 AM Page 61 mac76 mac76:385_reb: Chapter 2 Water 61 mated by measuring the concentrations at equilibrium. Kw  [H
][OH]  [H
]2 For reasons beyond the scope of this discussion, equi- Solving for [H
] gives librium constants are dimensionless. Nonetheless, we have generally retained the concentration units (M) in [H
] 
 Kw 
  1 1014 M