《生物化学原理》英文版 chapter 5 PROTEIN FUNCTION

8885ac05157-1898/12/038:55 AM Page157mac78mac78:385p chapter PROTEIN FUNCTION 5.1 Reversible Binding of a Protein to a Ligand: proteins interact with other molecules and how their in- Oxygen-Binding Proteins 158 teractions are related to dynamic protein structure. The 5.2 Complementary Interactions between Proteins importance of molecular interactions to a proteins func and Ligands: The Immune System and tion can hardly be overemphasized. In Chapter 4, we saw Immunoglobulins 174 that the function of fibrous proteins as structural ele- 5.3 Protein Interactions Modulated by Chemical Energy ments of cells and tissues depends on stable, long-term Actin, Myosin, and Molecular Motors 182 quaternary interactions between identical polypeptide chains. As we shall see in this chapter, the functions of many other proteins involve interactions with a variety have occasionally seen in almost dried blood, placed of different molecules. Most of these interactions are between glass plates in a desiccator, rectangula fleeting, though they may be the basis of complex phys- iological processes such as oxygen transport, immune crystalline structures, which under the microscope had function, and muscle contraction-the topics we exam- sharp edges and were bright red ine in detail in this chapter. The proteins that carry out -Friedrich Ludwig Hunefeld, Der Chemismus in these processes illustrate the following key principles of der thierischen Organisation, 1840 protein function, some of which will be familiar from the (one of the first observations of hemoglobin) chapte The functions of many proteins involve the Since the proteins participate in one way or another in all reversible binding of other molecules. A molecule chemical processes in the living organism, one may bound reversibly by a protein is called a ligand expect highly significant information for biological A ligand may be any kind of molecule, including chemistry from the elucidation of their structure and their another protein. The transient nature of protein- transformations ligand interactions is critical to life, allowing an -Emil Fischer article in berichte der deutschen organism to respond rapidly and reversibly to changing environmental and metabolic chemischen gesellschaft zu berlin, 1906 A ligand binds at a site on the protein called the binding site, which is complementary to the nowing the three-dimensional structure of a protein ligand in size, shape, charge, and hydrophobic or an important part of understanding how the pro- hydrophilic character. Furthermore, the interaction tein functions However the structure shown in two di is specific: the protein can discriminate among the mensions on a page is deceptively static. Proteins are thousands of different molecules in its environment dynamic molecules whose functions almost invariably and selectively bind only one or a few. a given depend on interactions with other molecules, and these protein may have separate binding sites for several interactions are affected in physiologically important different ligands. These specific molecular ways by sometimes subtle, sometimes striking changes interactions are crucial in maintaining the high in protein conformation. In this chapter, we explore how degree of order in a living system. (This discussion

chapter PROTEIN FUNCTION 5.1 Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins 158 5.2 Complementary Interactions between Proteins and Ligands: The Immune System and Immunoglobulins 174 5.3 Protein Interactions Modulated by Chemical Energy: Actin, Myosin, and Molecular Motors 182 I have occasionally seen in almost dried blood, placed between glass plates in a desiccator, rectangular crystalline structures, which under the microscope had sharp edges and were bright red. —Friedrich Ludwig Hünefeld, Der Chemismus in der thierischen Organisation, 1840 (one of the first observations of hemoglobin) Since the proteins participate in one way or another in all chemical processes in the living organism, one may expect highly significant information for biological chemistry from the elucidation of their structure and their transformations. —Emil Fischer, article in Berichte der deutschen chemischen Gesellschaft zu Berlin, 1906 5 Knowing the three-dimensional structure of a protein is an important part of understanding how the pro￾tein functions. However, the structure shown in two di￾mensions on a page is deceptively static. Proteins are dynamic molecules whose functions almost invariably depend on interactions with other molecules, and these interactions are affected in physiologically important ways by sometimes subtle, sometimes striking changes in protein conformation. In this chapter, we explore how proteins interact with other molecules and how their in￾teractions are related to dynamic protein structure. The importance of molecular interactions to a protein’s func￾tion can hardly be overemphasized. In Chapter 4, we saw that the function of fibrous proteins as structural ele￾ments of cells and tissues depends on stable, long-term quaternary interactions between identical polypeptide chains. As we shall see in this chapter, the functions of many other proteins involve interactions with a variety of different molecules. Most of these interactions are fleeting, though they may be the basis of complex phys￾iological processes such as oxygen transport, immune function, and muscle contraction—the topics we exam￾ine in detail in this chapter. The proteins that carry out these processes illustrate the following key principles of protein function, some of which will be familiar from the previous chapter: The functions of many proteins involve the reversible binding of other molecules. A molecule bound reversibly by a protein is called a ligand. A ligand may be any kind of molecule, including another protein. The transient nature of protein￾ligand interactions is critical to life, allowing an organism to respond rapidly and reversibly to changing environmental and metabolic circumstances. A ligand binds at a site on the protein called the binding site, which is complementary to the ligand in size, shape, charge, and hydrophobic or hydrophilic character. Furthermore, the interaction is specific: the protein can discriminate among the thousands of different molecules in its environment and selectively bind only one or a few. A given protein may have separate binding sites for several different ligands. These specific molecular interactions are crucial in maintaining the high degree of order in a living system. (This discussion 157 8885d_c05_157-189 8/12/03 8:55 AM Page 157 mac78 mac78:385_REB:

8885dc05157-1898/12/038:55 AM Page158mac78mac78:385 158 Part I Structure and Catalysis excludes the binding of water, which may interact molecules illustrate almost every aspect of that most weakly and nonspecifically with many parts of central of biochemical processes: the reversible binding protein. In Chapter 6, we consider water as a of a ligand to a protein. This classic model of protein specific ligand for many enzymes. function tells us a great deal about how proteins work Proteins are flexible. Changes in conformation 6 Oxygen-Binding Proteins--Myoglobin: Oxygen Storage may be subtle, reflecting molecular vibrations and Oxygen Can Be Bound to a Heme Prosthetic Group small movements of amino acid residues throughout the protein. a protein flexing in this Oxygen is poorly soluble in aqueous solutions (see Table way is sometimes said to"breathe. Changes in 2-3)and cannot be carried to tissues in sufficient quan conformation may also be quite dramatic, with tity if it is simply dissolved in blood serum. Diffusion of major segments of the protein structure moving oxygen through tissues is also ineffective over distances as much as several nanometers. Specific greater than a few millimeters. The evolution of larger onformational changes are frequently essential to multicellular animals depended on the evolution of pro- a proteins function. teins that could transport and store oxygen. However, The binding of a protein and ligand is often none of the amino acid side chains in proteins is suited for the reversible binding of oxygen molecules. This role coupled to a conformational change in the protein is filled by certain transition metals, among them iron that makes the binding site more complementary to the ligand, permitting tighter binding. The and copper, that have a strong tendency to bind oxy gen. Multicellular organisms exploit the properties of structural adaptation that occurs between proten metals, most commonly iron, for oxygen transport. How and ligand is called induced fit. ever, free iron promotes the formation of highly reac In a multisubunit protein, a conformational tive oxygen species such as hydroxyl radicals that can change in one subunit often affects the damage DNA and other macromolecules. Iron used in conformation of other subunits cells is therefore bound in forms that sequester it and/or Interactions between ligands and proteins may be make it less reactive In multicellular organisms--espe- regulated, usually through specific interactions cially those in which iron, in its oxygen-carrying capac with one or more additional ligands. These other ty, must be transported over large distances--iron is of- igands may cause conformational changes in the ten incorporated into a protein-bound prosthetic group protein that affect the binding of the first ligand called heme.(Recall from Chapter 3 that a prosthetic group is a compound permanently associated with a pro- Enzymes represent a special case of protein func tein that contributes to the proteins function.) Heme (or haen) consists of a complex organic ring tion. Enzymes bind and chemically transform other mol- structure, protoporphyrin, to which is bound a single ecules--they catalyze reactions. The molecules acted upon by enzymes are called reaction substrates rather iron atom in its ferrous(Fe-s) state(Fig 5-1). The iron than ligands, and the ligand-binding site is called the atom has six coordination bonds, four to nitrogen atoms that are part of the flat porphyrin ring system and catalytic site or active site. In this chapter we two perpendicular to the porphyrin. The coordinated emphasize the noncatalytic functions of proteins. In Chapter 6 we consider catalysis by enzymes, a central nitrogen atoms(which have an electron-donating char topic in biochemistry. You will see that the themes of acter) help prevent conversion of the heme iron to the this chapter--binding, specificity, and conformational ferric(Fe t )state. Iron in the Fet state binds oxygen change-are continued in the next chapter, with the reversibly; in the Fe state it does not bind oxygen. added element of proteins acting as reactants in chem- Heme is found in a number of oxygen-transporting ical transformations proteins, as well as in some proteins, such as the cytochromes, that participate in oxidation-reduction (electron-transfer) reactions(Chapter 19) In free heme molecules (heme not bound to pro- 5.1 Reversible Binding of a Protein tein), reaction of oxygen at one of the two"open"CO- to a Ligand: Oxygen-Binding Proteins ordination bonds of iron(perpendicular to the plane of the porphyrin molecule, above and below) can result Myoglobin and hemoglobin may be the most-studied and in irreversible conversion of Fe-t to Fe. In heme- best-understood proteins. They were the first proteins containing proteins, this reaction is prevented by se- for which three-dimensional structures were deter- questering of the heme deep within the protein struc mined, and our current understanding of myoglobin and ture where access to the two open coordination bonds hemoglobin is garnered from the work of thousands of is restricted. One of these two coordination bonds is oc- biochemists over several decades. Most important, these cupid by a side-chain nitrogen of a His residue. The

excludes the binding of water, which may interact weakly and nonspecifically with many parts of a protein. In Chapter 6, we consider water as a specific ligand for many enzymes.) Proteins are flexible. Changes in conformation may be subtle, reflecting molecular vibrations and small movements of amino acid residues throughout the protein. A protein flexing in this way is sometimes said to “breathe.” Changes in conformation may also be quite dramatic, with major segments of the protein structure moving as much as several nanometers. Specific conformational changes are frequently essential to a protein’s function. The binding of a protein and ligand is often coupled to a conformational change in the protein that makes the binding site more complementary to the ligand, permitting tighter binding. The structural adaptation that occurs between protein and ligand is called induced fit. In a multisubunit protein, a conformational change in one subunit often affects the conformation of other subunits. Interactions between ligands and proteins may be regulated, usually through specific interactions with one or more additional ligands. These other ligands may cause conformational changes in the protein that affect the binding of the first ligand. Enzymes represent a special case of protein func￾tion. Enzymes bind and chemically transform other mol￾ecules—they catalyze reactions. The molecules acted upon by enzymes are called reaction substrates rather than ligands, and the ligand-binding site is called the catalytic site or active site. In this chapter we emphasize the noncatalytic functions of proteins. In Chapter 6 we consider catalysis by enzymes, a central topic in biochemistry. You will see that the themes of this chapter—binding, specificity, and conformational change—are continued in the next chapter, with the added element of proteins acting as reactants in chem￾ical transformations. 5.1 Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins Myoglobin and hemoglobin may be the most-studied and best-understood proteins. They were the first proteins for which three-dimensional structures were deter￾mined, and our current understanding of myoglobin and hemoglobin is garnered from the work of thousands of biochemists over several decades. Most important, these molecules illustrate almost every aspect of that most central of biochemical processes: the reversible binding of a ligand to a protein. This classic model of protein function tells us a great deal about how proteins work. Oxygen-Binding Proteins—Myoglobin: Oxygen Storage Oxygen Can Be Bound to a Heme Prosthetic Group Oxygen is poorly soluble in aqueous solutions (see Table 2–3) and cannot be carried to tissues in sufficient quan￾tity if it is simply dissolved in blood serum. Diffusion of oxygen through tissues is also ineffective over distances greater than a few millimeters. The evolution of larger, multicellular animals depended on the evolution of pro￾teins that could transport and store oxygen. However, none of the amino acid side chains in proteins is suited for the reversible binding of oxygen molecules. This role is filled by certain transition metals, among them iron and copper, that have a strong tendency to bind oxy￾gen. Multicellular organisms exploit the properties of metals, most commonly iron, for oxygen transport. How￾ever, free iron promotes the formation of highly reac￾tive oxygen species such as hydroxyl radicals that can damage DNA and other macromolecules. Iron used in cells is therefore bound in forms that sequester it and/or make it less reactive. In multicellular organisms—espe￾cially those in which iron, in its oxygen-carrying capac￾ity, must be transported over large distances—iron is of￾ten incorporated into a protein-bound prosthetic group called heme. (Recall from Chapter 3 that a prosthetic group is a compound permanently associated with a pro￾tein that contributes to the protein’s function.) Heme (or haem) consists of a complex organic ring structure, protoporphyrin, to which is bound a single iron atom in its ferrous (Fe2
) state (Fig. 5–1). The iron atom has six coordination bonds, four to nitrogen atoms that are part of the flat porphyrin ring system and two perpendicular to the porphyrin. The coordinated nitrogen atoms (which have an electron-donating char￾acter) help prevent conversion of the heme iron to the ferric (Fe3
) state. Iron in the Fe2
state binds oxygen reversibly; in the Fe3
state it does not bind oxygen. Heme is found in a number of oxygen-transporting proteins, as well as in some proteins, such as the cytochromes, that participate in oxidation-reduction (electron-transfer) reactions (Chapter 19). In free heme molecules (heme not bound to pro￾tein), reaction of oxygen at one of the two “open” co￾ordination bonds of iron (perpendicular to the plane of the porphyrin molecule, above and below) can result in irreversible conversion of Fe2
to Fe3
. In heme￾containing proteins, this reaction is prevented by se￾questering of the heme deep within the protein struc￾ture where access to the two open coordination bonds is restricted. One of these two coordination bonds is oc￾cupied by a side-chain nitrogen of a His residue. The 158 Part I Structure and Catalysis 8885d_c05_157-189 8/12/03 8:55 AM Page 158 mac78 mac78:385_REB:

8885ac05157-1898/12/038:55 AM Page159mac78mac78:385 Protein Function CH CHI Fe- ch CH CH CH FIGURE 5-1 Heme. The heme group is present in myoglobin, hemo- role rings linked by methene bridges, with substitutions at one or more globin, and many other proteins, designated heme proteins. Heme of the positions denoted X. (b, c) Two representations of heme(De- onsists of a complex organic ring structure, protoporphyrin IX, to rived from PDB ID 1CCR The iron atom of heme has six coordina- hich is bound an iron atom in its ferrous(Fe2+)state. (a) Porphyrins tion bonds: four in the plane of, and bonded to, the flat porphyrin ring of which protoporphyrin IX is only one example, consist of four pyr- stem,and(d) two perpendicular to it. other is the binding site for molecular oxygen(O2)(Fig Myoglobin Has a Single Binding Site for Oxygen 5-2). When oxygen binds, the electronic properties of heme iron change; this accounts for the change in color Myoglobin(Mr 16, 700; abbreviated Mb) is a relatively from the dark purple of oxygen-depleted venous blood simple oxygen-binding protein found in almost all mam- to the bright red of oxygen-rich arterial blood. Some mals, primarily in muscle tissue. As a transport protein, it facilitates oxygen diffusion in muscle. Myoglobin is tric oxide (NO), coordinate to heme iron with greater particularly abundant in the muscles of diving mammals affinity than does O, When a molecule of co is bound such as seals and whales, where it also has an oxygen- to heme, O2 is excluded, which is why Co is highly toxic storage function for prolonged excursions undersea to aerobic organisms(a topic explored later, in Box Proteins very similar to myoglobin are widely distrib- 1). By surrounding and sequestering heme, oxygen uted, occurring even in some single-celled organisms binding proteins regulate the access of CO and other Myoglobin is a single polypeptide of 153 amino acid small molecules to the heme iron residues with one molecule of heme. It is typical of the family of proteins called globins, all of which have sim- ilar primary and tertiary structures. The polypeptide is made up of eight a-helical segments connected by bends Edge view (Fig. 5-3). About 78% of the amino acid residues in the protein are found in these a helices. Any detailed discussion of protein function in- evitably involves protein structure. To facilitate our treatment of myoglobin, we first introduce some struc tural conventions peculiar to globins. As seen in Figure 5-3, the helical segments are named A through H. An individual amino acid residue is designated either by its Histidine Plane of position in the amino acid sequence or by its location residue porphyrin within the sequence of a particular a-helical segment ring system For example, the His residue coordinated to the heme FIGURE 5-2 The heme group viewed from the side. This view shows in myoglobin, His(the 93rd amino acid residue from the two coordination bonds to Fe2+ perpendicular to the porphyri the amino-terminal end of the myoglobin polypeptide ring system. One of these two bonds is occupied by a His residu sequence), is also called His F8(the Sth residue in a sometimes called the proximal His. The other bond is the binding site helix F). The bends in the structure are designated AB for oxygen. The remaining four coordination bonds are in the plane CD, EF, FG, and so forth, reflecting the a-helical seg of, and bonded to, the flat porphyrin ring system ments they connect

other is the binding site for molecular oxygen (O2) (Fig. 5–2). When oxygen binds, the electronic properties of heme iron change; this accounts for the change in color from the dark purple of oxygen-depleted venous blood to the bright red of oxygen-rich arterial blood. Some small molecules, such as carbon monoxide (CO) and ni￾tric oxide (NO), coordinate to heme iron with greater affinity than does O2. When a molecule of CO is bound to heme, O2 is excluded, which is why CO is highly toxic to aerobic organisms (a topic explored later, in Box 5–1). By surrounding and sequestering heme, oxygen￾binding proteins regulate the access of CO and other small molecules to the heme iron. Myoglobin Has a Single Binding Site for Oxygen Myoglobin (Mr 16,700; abbreviated Mb) is a relatively simple oxygen-binding protein found in almost all mam￾mals, primarily in muscle tissue. As a transport protein, it facilitates oxygen diffusion in muscle. Myoglobin is particularly abundant in the muscles of diving mammals such as seals and whales, where it also has an oxygen￾storage function for prolonged excursions undersea. Proteins very similar to myoglobin are widely distrib￾uted, occurring even in some single-celled organisms. Myoglobin is a single polypeptide of 153 amino acid residues with one molecule of heme. It is typical of the family of proteins called globins, all of which have sim￾ilar primary and tertiary structures. The polypeptide is made up of eight
-helical segments connected by bends (Fig. 5–3). About 78% of the amino acid residues in the protein are found in these
helices. Any detailed discussion of protein function in￾evitably involves protein structure. To facilitate our treatment of myoglobin, we first introduce some struc￾tural conventions peculiar to globins. As seen in Figure 5–3, the helical segments are named A through H. An individual amino acid residue is designated either by its position in the amino acid sequence or by its location within the sequence of a particular
-helical segment. For example, the His residue coordinated to the heme in myoglobin, His93 (the 93rd amino acid residue from the amino-terminal end of the myoglobin polypeptide sequence), is also called His F8 (the 8th residue in
helix F). The bends in the structure are designated AB, CD, EF, FG, and so forth, reflecting the
-helical seg￾ments they connect. Chapter 5 Protein Function 159 O C O O Fe
CH3 CH N
CH2 CH2 CH2 CH2 CH2 C H3 C H3 CH3 CH CH CH CH CH O C C C C C C C C C C C C C C C N N N CH2 C (b) C (a) NH X N HN N X X X X X X X (c) (d) Fe FIGURE 5–1 Heme. The heme group is present in myoglobin, hemo￾globin, and many other proteins, designated heme proteins. Heme consists of a complex organic ring structure, protoporphyrin IX, to which is bound an iron atom in its ferrous (Fe2
) state. (a) Porphyrins, of which protoporphyrin IX is only one example, consist of four pyr￾role rings linked by methene bridges, with substitutions at one or more of the positions denoted X. (b, c) Two representations of heme. (De￾rived from PDB ID 1CCR.) The iron atom of heme has six coordina￾tion bonds: four in the plane of, and bonded to, the flat porphyrin ring system, and (d) two perpendicular to it. FIGURE 5–2 The heme group viewed from the side. This view shows the two coordination bonds to Fe2
perpendicular to the porphyrin ring system. One of these two bonds is occupied by a His residue, sometimes called the proximal His. The other bond is the binding site for oxygen. The remaining four coordination bonds are in the plane of, and bonded to, the flat porphyrin ring system. HN CH2 C CH Edge view ring system residue C N Fe O2 Histidine Plane of porphyrin H 8885d_c05_157-189 8/12/03 8:55 AM Page 159 mac78 mac78:385_REB:

8885dc05157-1898/12/038:55 AM Page160mac78mac78:385 160 Part I Structure and Catalysis a higher affinity of the ligand for the protein. a re- arrangement of Equation 5-2 shows that the ratio of bound to free protein is directly proportional to the con KaLLI When the concentration of the ligand is much greater than the concentration of ligand-binding sites, the binding of the ligand by the protein does not apprecia- bly change the concentration of free (unbound)li- gand-that is, L remains constant. This condition is broadly applicable to most ligands that bind to proteins in cells and simplifies our description of the binding equilibrium. We can now consider the binding equilibrium from the standpoint of the fraction, e(theta), of ligand binding sites on the protein that are occupied by ligand FIGURE 5-3 The structure of myoglobin (PDB ID 1MBO) The eight IPL (5-4) a-helical segments (shown here as cylinders) are labeled A through H. Nonhelical residues in the bends that connect them are labeled Substituting KalLJP for [PL(see Eqn 5-3)and re- AB,CD,EF, and so forth, indicating the segments they interconnect. arranging terms gives A few bends, including BC and DE, are abrupt and do not contain any residues; these are not normally labeled. (The short segment vis- KaLLJPI ble between D and E is an artifact of the computer representation The heme is bound in a pocket made up largely of the E and F he. although amino acid residues from other segments of the pro The value of Ka can be determined from a plot of e ver also participate. sus the concentration of free ligand, L(Fig. 5-4a). Any equation of the form =y/(y +2) describes a hyper bola, and 0 is thus found to be a hyperbolic function of Protein-Ligand Interactions Can Be L. The fraction of ligand-binding sites occupied ap- Described Quantitatively proaches saturation asymptotically as l increases. The L at which half of the available ligand-binding sites are The function of myoglobin depends on the proteins abil- occupied (at 0=0.5) corresponds to 1/K ity not only to bind oxygen but also to release it when It is more common (and intuitively simpler), how- and where it is needed. Function in biochemistry often ever, to consider the dissociation constant, Kd, which revolves around a reversible protein-ligand interaction is the reciprocal of Ka(Kd= 1/Ka) and is given in units of this type. A quantitative description of this interac- of molar concentration(M). Ka is the equilibrium con- tion is therefore a central part of many biochemical in- stant for the release of ligand. The relevant expressions vestigations change to In general, the reversible binding of a protein(P) to a ligand () can be described by a simple equilib- rium expression: P+L (5-1) The reaction is characterized by an equilibrium con- stant, K. such that (5-2) When L is equal to Kd, half of the ligand-binding sites are occupied. As falls below Kd, progressively less of The term Ka is an association constant (not to be the protein has ligand bound to it. In order for 90%of confused with the Ka that denotes an acid dissociation the available ligand-binding sites to be occupied, L onstant; p. 63). The association constant provides a must be nine times greater than Kd measure of the affinity of the ligand L for the protein. In practice, Kd is used much more often than Ka to Ka has units of M; a higher value of Ka corresponds to express the affinity of a protein for a ligand. Note that

Protein-Ligand Interactions Can Be Described Quantitatively The function of myoglobin depends on the protein’s abil￾ity not only to bind oxygen but also to release it when and where it is needed. Function in biochemistry often revolves around a reversible protein-ligand interaction of this type. A quantitative description of this interac￾tion is therefore a central part of many biochemical in￾vestigations. In general, the reversible binding of a protein (P) to a ligand (L) can be described by a simple equilib￾rium expression: P
L PL (5–1) The reaction is characterized by an equilibrium con￾stant, Ka, such that Ka [ [ P P ] L [L ] ] (5–2) The term Ka is an association constant (not to be confused with the Ka that denotes an acid dissociation constant; p. 63). The association constant provides a measure of the affinity of the ligand L for the protein. Ka has units of M1 ; a higher value of Ka corresponds to yz a higher affinity of the ligand for the protein. A re￾arrangement of Equation 5–2 shows that the ratio of bound to free protein is directly proportional to the con￾centration of free ligand: Ka[L]  [P [P L ] ]  (5–3) When the concentration of the ligand is much greater than the concentration of ligand-binding sites, the binding of the ligand by the protein does not apprecia￾bly change the concentration of free (unbound) li￾gand—that is, [L] remains constant. This condition is broadly applicable to most ligands that bind to proteins in cells and simplifies our description of the binding equilibrium. We can now consider the binding equilibrium from the standpoint of the fraction, (theta), of ligand￾binding sites on the protein that are occupied by ligand: [PL [P ]
L] [P] (5–4) Substituting Ka[L][P] for [PL] (see Eqn 5–3) and re￾arranging terms gives Ka[ K L a ][ [ P L ] ][
P] [P] Ka K [L a[ ] L
] 1 (5–5) The value of Ka can be determined from a plot of ver￾sus the concentration of free ligand, [L] (Fig. 5–4a). Any equation of the form x y/(y
z) describes a hyper￾bola, and is thus found to be a hyperbolic function of [L]. The fraction of ligand-binding sites occupied ap￾proaches saturation asymptotically as [L] increases. The [L] at which half of the available ligand-binding sites are occupied (at 0.5) corresponds to 1/Ka. It is more common (and intuitively simpler), how￾ever, to consider the dissociation constant, Kd, which is the reciprocal of Ka (Kd 1/Ka) and is given in units of molar concentration (M). Kd is the equilibrium con￾stant for the release of ligand. The relevant expressions change to Kd  [P [P ][ L L ] ]  (5–6) [PL]  [P K ][ d L]  (5–7) [L] [
L] Kd (5–8) When [L] is equal to Kd, half of the ligand-binding sites are occupied. As [L] falls below Kd, progressively less of the protein has ligand bound to it. In order for 90% of the available ligand-binding sites to be occupied, [L] must be nine times greater than Kd. In practice, Kd is used much more often than Ka to express the affinity of a protein for a ligand. Note that  [L] [L]
K 1 a   binding sites occupied total binding sites 160 Part I Structure and Catalysis A EF F H FG C CD D B G E GH AB FIGURE 5–3 The structure of myoglobin. (PDB ID 1MBO) The eight
-helical segments (shown here as cylinders) are labeled A through H. Nonhelical residues in the bends that connect them are labeled AB, CD, EF, and so forth, indicating the segments they interconnect. A few bends, including BC and DE, are abrupt and do not contain any residues; these are not normally labeled. (The short segment vis￾ible between D and E is an artifact of the computer representation.) The heme is bound in a pocket made up largely of the E and F he￾lices, although amino acid residues from other segments of the pro￾tein also participate. 8885d_c05_157-189 8/12/03 8:55 AM Page 160 mac78 mac78:385_REB:

8885ac05157-1898/12/038:55 AM Page161mac78mac78:385p Protein function 161 1.0 1.0 00.5 0.5 (arbitrary units) (b) pO2(kPa) FIGURE 5-4 Graphical representations of ligand binding. The frac- or Kd. The curve has a horizontal asymptote at 0=1 and a vertical tion of ligand-binding sites occupied, e, is plotted against the con- asymptote (not shown)at [L]=-1/Ka (b)A curve describing the bind centration of free ligand. Both curves are rectangular hyperbolas. ing of oxygen to myoglobin. The partial pressure of O2 in the air above (a)A hypothetical binding curve for a ligand L. The [L] at which half the solution is expressed in kilopascals(kPa). Oxygen binds tightly to of the available ligand-binding sites are occupied is equivalent to 1/K. myoglobin, with a Pso of only 0.26 kPa a lower value of Kd corresponds to a higher affinity of As for any ligand, Ka is equal to the [oa] at which half igand for the protein. The mathematics can be reduced of the available ligand-binding sites are occupied, or to simple statements: Kd is equivalent to the molar con- [O.s. Equation 5-9 thus becomes centration of ligand at which half of the available ligand binding sites are occupied. At this point, the protein is (5-10) said to have reached half-saturation with respect to lig. nd binding. The more tightly a protein binds a ligand, In experiments using oxygen as a ligand, it is the par the lower the concentration of ligand required for half tial pressure of oxygen in the gas phase above the the binding sites to be occupied, and thus the lower the solution, pO,, that is varied, because this is easier to value of Kd. Some representative dissociation constants measure than the concentration of oxygen dissolved in are given in Table 5-1 the solution The concentration of a volatile substance The binding of oxygen to myoglobin follows the pat in solution is always proportional to the local partial terns discussed above. However, because oxygen is a pressure of the gas. So, if we define the partial pressure gas, we must make some minor adjustments to the equa- of oxygen at [ojO.s as Pso, substitution in Equation 5-10 tions so that laboratory experiments can be carried out gives more conveniently. We first substitute the concentration 02 of dissolved oxygen for L in Equation 5-8 to give (5-11) a binding curve for myoglobin that relates e to pOe is shown in Figure 5-4b TABLE 5-1 Some Protein Dissociation Constants Prote Ligand Kd(M) Avidin(egg white Biotin 1×10 Insulin receptor(human) Insulin 1×10 gp41(HIV-1 surface protein) 4 Nickel-binding protein(E. co) 1×10-7 3×10-6 rticular solution cond tions under which it was measured. Ke values for a protein-ligand interaction I be altered, sometimes by several orders of magnitude, by changes in the solutions salt concentration, pH, or other variables. ' interaction of avidin with biotin, an enzyme cofactor, is among the strongest noncovalent biochemical interactions known. "This immunoglobulin w and the K repo should not be considered characteristic of all immunoglobulins Calmodulin has four binding sites for calum. The values shown reflect the highest- and lowest-affinity bind ng sites observed in one set of measurements

a lower value of Kd corresponds to a higher affinity of ligand for the protein. The mathematics can be reduced to simple statements: Kd is equivalent to the molar con￾centration of ligand at which half of the available ligand￾binding sites are occupied. At this point, the protein is said to have reached half-saturation with respect to lig￾and binding. The more tightly a protein binds a ligand, the lower the concentration of ligand required for half the binding sites to be occupied, and thus the lower the value of Kd. Some representative dissociation constants are given in Table 5–1. The binding of oxygen to myoglobin follows the pat￾terns discussed above. However, because oxygen is a gas, we must make some minor adjustments to the equa￾tions so that laboratory experiments can be carried out more conveniently. We first substitute the concentration of dissolved oxygen for [L] in Equation 5–8 to give [O2 [ ] O
2] Kd (5–9) As for any ligand, Kd is equal to the [O2] at which half of the available ligand-binding sites are occupied, or [O2]0.5. Equation 5–9 thus becomes [O2]
[O [ 2 O ]  2]0.5 (5–10) In experiments using oxygen as a ligand, it is the par￾tial pressure of oxygen in the gas phase above the solution, pO2, that is varied, because this is easier to measure than the concentration of oxygen dissolved in the solution. The concentration of a volatile substance in solution is always proportional to the local partial pressure of the gas. So, if we define the partial pressure of oxygen at [O2]0.5 as P50, substitution in Equation 5–10 gives pO2 pO
2 P50 (5–11) A binding curve for myoglobin that relates to pO2 is shown in Figure 5–4b. Chapter 5 Protein Function 161 1.0 0.5 0 v P50 5 10 (b) pO2 (kPa) 1.0 0.5 0 v 5 (a) Kd 10 [L] (arbitrary units) FIGURE 5–4 Graphical representations of ligand binding. The frac￾tion of ligand-binding sites occupied, , is plotted against the con￾centration of free ligand. Both curves are rectangular hyperbolas. (a) A hypothetical binding curve for a ligand L. The [L] at which half of the available ligand-binding sites are occupied is equivalent to 1/Ka, or Kd. The curve has a horizontal asymptote at 1 and a vertical asymptote (not shown) at [L] 1/Ka. (b) A curve describing the bind￾ing of oxygen to myoglobin. The partial pressure of O2 in the air above the solution is expressed in kilopascals (kPa). Oxygen binds tightly to myoglobin, with a P50 of only 0.26 kPa. TABLE 5–1 Some Protein Dissociation Constants Protein Ligand Kd (M)* Avidin (egg white)† Biotin 1  1015 Insulin receptor (human) Insulin 1  1010 Anti-HIV immunoglobulin (human)‡ gp41 (HIV-1 surface protein) 4  1010 Nickel-binding protein (E. coli) Ni2
1  107 Calmodulin (rat)§ Ca2
3  106 2  105 *A reported dissociation constant is valid only for the particular solution conditions under which it was measured. Kd values for a protein-ligand interaction can be altered, sometimes by several orders of magnitude, by changes in the solution’s salt concentration, pH, or other variables. † Interaction of avidin with biotin, an enzyme cofactor, is among the strongest noncovalent biochemical interactions known. ‡ This immunoglobulin was isolated as part of an effort to develop a vaccine against HIV. Immunoglobulins (described later in the chapter) are highly variable, and the Kd reported here should not be considered characteristic of all immunoglobulins. § Calmodulin has four binding sites for calcium. The values shown reflect the highest- and lowest-affinity binding sites observed in one set of measurements. 8885d_c05_157-189 8/12/03 8:55 AM Page 161 mac78 mac78:385_REB:

8885ac05_157-1898/12/038:55 AM Page162mac78mac78:385 162 Part I Structure and Catalysis Protein Structure Affects How Ligands Bind The binding of a ligand to a protein is rarely as simple as the above equations would suggest. The interaction Fe Is greatly affected by protein structure and is often ac- (a) companied by conformational changes. For example the specificity with which heme binds its various ligands is altered when the heme is a component of myoglobin Carbon monoxide binds to free heme molecules more than 20,000 times better than does O2(that is, the Kd or Pso for Co binding to free heme is more than 20,000 times lower than that for O2), but it binds only about 200 times better when the heme is bound in myoglobin The difference may be partly explained by steric hin- Phe cD1 drance. When Oe binds to free heme, the axis of the oxy- gen molecule is positioned at an angle to the Fe-0 bond val ell( (Fig. 5-5a). In contrast, when Co binds to free heme the Fe, C, and o atoms lie in a straight line (Fig. 5-5b) In both cases, the binding reflects the geometry of hy brid orbitals in each ligand In myoglobin, His(His E7 on the Oo-binding side of the heme, is too far away to coordinate with the heme iron but it does interact with a ligand bound to heme. This residue, called the distal His, does not affect the binding of O2(Fig. 5-5c)but His F may preclude the linear binding of CO, providing one explanation for the diminished binding of Co to heme in myoglobin(and hemoglobin). A reduction in CO bind- ing is physiologically important, because Co is a lot level byproduct of cellular metabolism. Other factors not yet well-defined, also seem to modulate the inter- FIGURE 5-5 Steric effects on the binding of ligands to the heme of action of heme with CO in these proteins myoglobin. (a) Oxygen binds to heme with the O2 axis at an angle, The binding of O2 to the heme in myoglobin also de- a binding conformation readily accommodated by myoglobin. (b)Car- pends on molecular motions, or "breathing, "in the pro- bon monoxide binds to free heme with the CO axis perpendicular tein structure. The heme molecule is deeply buried in the plane of the porphyrin ring. When binding to the heme in myo- the folded polypeptide, with no direct path for oxyge globin, CO is forced to adopt a slight angle because the perpendicu to move from the surrounding solution to the ligand lar arrangement is sterically blocked by His E7, the distal His. This ef- binding site. If the protein were rigid, O, could not en- fect weakens the binding of co to myoglobin. (c) Another view ter or leave the heme pocket at a measurable rate How- ever, rapid molecular flexing of the amino acid side acid residues around the heme of myoglobin. The bound Oz is hy chains produces transient cavities in the protein struc- drogen-bonded to the distal His, His E7(His), further facilitating the ture, and Oe evidently makes its way in and out by mov- ing through these cavities. Computer simulations of rapid structural fluctuations in myoglobin suggest that there are many such pathways. One major route is pro- the maturation process, the stem cell produces daugh- vided by rotation of the side chain of the distal His ter cells that form large amounts of hemoglobin and then (His), which occurs on a nanosecond (10s) time lose their intracellular organelles--nucleus, mitochon- scale. Even subtle conformational changes can be criti dria, and endoplasmic reticulum Erythrocytes are thus cal for protein activity incomplete, vestigial cells, unable to reproduce and, in humans, destined to survive for only about 120 days. Oxygen Is Transported in Blood by Hemoglobin Their main function is to carry hemoglobin, which is dis solved in the cytosol at a very high concentration (-34% 9 Oxygen-Binding Proteins-Hemoglobin: Oxygen Transport by weight) Nearly all the oxygen carried by whole blood in animals In arterial blood passing from the lungs through the is bound and transported by hemoglobin in erythrocytes heart to the peripheral tissues, hemoglobin is about 96% (red blood cells). Normal human erythrocytes are small saturated with oxygen. In the venous blood returning to (6 to 9 um in diameter), biconcave disks. They are formed the heart, hemoglobin is only about 64% saturated. Thus from precursor stem cells called hemocytoblasts. In each 100 mL of blood passing through a tissue releases

Protein Structure Affects How Ligands Bind The binding of a ligand to a protein is rarely as simple as the above equations would suggest. The interaction is greatly affected by protein structure and is often ac￾companied by conformational changes. For example, the specificity with which heme binds its various ligands is altered when the heme is a component of myoglobin. Carbon monoxide binds to free heme molecules more than 20,000 times better than does O2 (that is, the Kd or P50 for CO binding to free heme is more than 20,000 times lower than that for O2), but it binds only about 200 times better when the heme is bound in myoglobin. The difference may be partly explained by steric hin￾drance. When O2 binds to free heme, the axis of the oxy￾gen molecule is positioned at an angle to the FeOO bond (Fig. 5–5a). In contrast, when CO binds to free heme, the Fe, C, and O atoms lie in a straight line (Fig. 5–5b). In both cases, the binding reflects the geometry of hy￾brid orbitals in each ligand. In myoglobin, His64 (His E7), on the O2-binding side of the heme, is too far away to coordinate with the heme iron, but it does interact with a ligand bound to heme. This residue, called the distal His, does not affect the binding of O2 (Fig. 5–5c) but may preclude the linear binding of CO, providing one explanation for the diminished binding of CO to heme in myoglobin (and hemoglobin). A reduction in CO bind￾ing is physiologically important, because CO is a low￾level byproduct of cellular metabolism. Other factors, not yet well-defined, also seem to modulate the inter￾action of heme with CO in these proteins. The binding of O2 to the heme in myoglobin also de￾pends on molecular motions, or “breathing,” in the pro￾tein structure. The heme molecule is deeply buried in the folded polypeptide, with no direct path for oxygen to move from the surrounding solution to the ligand￾binding site. If the protein were rigid, O2 could not en￾ter or leave the heme pocket at a measurable rate. How￾ever, rapid molecular flexing of the amino acid side chains produces transient cavities in the protein struc￾ture, and O2 evidently makes its way in and out by mov￾ing through these cavities. Computer simulations of rapid structural fluctuations in myoglobin suggest that there are many such pathways. One major route is pro￾vided by rotation of the side chain of the distal His (His64), which occurs on a nanosecond (109 s) time scale. Even subtle conformational changes can be criti￾cal for protein activity. Oxygen Is Transported in Blood by Hemoglobin Oxygen-Binding Proteins—Hemoglobin: Oxygen Transport Nearly all the oxygen carried by whole blood in animals is bound and transported by hemoglobin in erythrocytes (red blood cells). Normal human erythrocytes are small (6 to 9 m in diameter), biconcave disks. They are formed from precursor stem cells called hemocytoblasts. In the maturation process, the stem cell produces daugh￾ter cells that form large amounts of hemoglobin and then lose their intracellular organelles—nucleus, mitochon￾dria, and endoplasmic reticulum. Erythrocytes are thus incomplete, vestigial cells, unable to reproduce and, in humans, destined to survive for only about 120 days. Their main function is to carry hemoglobin, which is dis￾solved in the cytosol at a very high concentration (~34% by weight). In arterial blood passing from the lungs through the heart to the peripheral tissues, hemoglobin is about 96% saturated with oxygen. In the venous blood returning to the heart, hemoglobin is only about 64% saturated. Thus, each 100 mL of blood passing through a tissue releases 162 Part I Structure and Catalysis FIGURE 5–5 Steric effects on the binding of ligands to the heme of myoglobin. (a) Oxygen binds to heme with the O2 axis at an angle, a binding conformation readily accommodated by myoglobin. (b) Car￾bon monoxide binds to free heme with the CO axis perpendicular to the plane of the porphyrin ring. When binding to the heme in myo￾globin, CO is forced to adopt a slight angle because the perpendicu￾lar arrangement is sterically blocked by His E7, the distal His. This ef￾fect weakens the binding of CO to myoglobin. (c) Another view (derived from PDB ID 1MBO), showing the arrangement of key amino acid residues around the heme of myoglobin. The bound O2 is hy￾drogen-bonded to the distal His, His E7 (His64), further facilitating the binding of O2. Phe CD1 His E7 His F8 (c) Fe H O2 Val E11 (a) O X A O O Fe A O J (b) O X A O O Fe A c C 8885d_c05_157-189 8/12/03 8:55 AM Page 162 mac78 mac78:385_REB:

8885ac05157-1898/12/038:55 AM Page163mac78mac78:385p Mb Hba Hb Mb Hbo HbB about one-third of the oxygen it carries, or 6.5 mL of O gas at atmospheric pressure and body temperature E D E Myoglobin, with its hyperbolic binding curve for oxygen (Fig. 5-4b), is relatively insensitive to small changes in the concentration of dissolved oxygen and LLL so functions well as an oxygen-storage protein. Hemo- globin, with its multiple subunits and O2-binding sites is better suited to oxygen transport. As we shall see, in- teractions between the subunits of a multimeric protein can permit a highly sensitive response to small changes GKvGAHAGEYGAEA LHcDKLHv in ligand concentration Interactions among the subunits in hemoglobin cause conformational changes that alter the affinity of the protein for oxygen. The modulation of oxygen binding allows the Oe-transport protein to re- spond to changes in oxygen demand by tissues Hemoglobin Subunits Are Structurally Similar to Myoglobin Hemoglobin (Mr 64, 500; abbreviated Hb) is rou LGNvLvcvLAHH spherical, with a diameter of nearly 5.5 nm. It is a tetrameric protein containing four heme prosthetic groups, one associated with each polypeptide chain. Adult hemoglobin contains two types of globin, two c chains(141 residues each) and two B chains (146 EFTP residues each). Although fewer than half of the amino acid residues in the polypeptide sequences of the a and B subunits are identical, the three-dimensional struc tures of the two types of subunits are very similar. Fur thermore, their structures are very similar to that of -G myoglobin(Fig. 5-6), even though the amino acid se- bers of the globin family of proteins. The helix-naming HE+ quences of the three polypeptides are identical at only 27 positions(Fig. 5-7). All three polypeptides are mem- convention described for myoglobin is also applied to the hemoglobin polypeptides, except that the a subunit lacks the short D helix. The heme-binding pocket made up largely of the e and f helices H26--L--- T Heme FIGURE 5-7 The amino acid sequences of whale myoglobin and the a and B chains of human hemoglobin Dashed lines mark helix bound. aries. To align the sequences optimally, short gaps must be introduced into both Hb sequences where a few amino acids are present in the mpared sequences. With the exception of the missing D helix in Hba, this alignment permits the use of the helix lettering convention that emphasizes the common positioning of amino acid residues that are identical in all three structures(shaded). Residues shaded in pink are conserved in all known globins. Note that the common helix letter- and-number designation for amino acids does not necessarily corre- spond to a common position in the linear sequence of amino acids Myoglobin B subunit of in the polypeptides. For example, the distal His residue is His E7 all three structures, but corresponds to His4, HisB, and Hissin the linear sequences of Mb, Hba, and HbB, respectively. Nonhelical FIGURE 5-6 A comparison of the structures of myoglobin( PDB Id residues at the amino and carboxyl termini, beyond the first(A) and MBO)and the B subunit of hemoglobin(derived from PDB ID 1 HGA). last(H)a-helical segments, are labeled NA and HC, respectively

about one-third of the oxygen it carries, or 6.5 mL of O2 gas at atmospheric pressure and body temperature. Myoglobin, with its hyperbolic binding curve for oxygen (Fig. 5–4b), is relatively insensitive to small changes in the concentration of dissolved oxygen and so functions well as an oxygen-storage protein. Hemo￾globin, with its multiple subunits and O2-binding sites, is better suited to oxygen transport. As we shall see, in￾teractions between the subunits of a multimeric protein can permit a highly sensitive response to small changes in ligand concentration. Interactions among the subunits in hemoglobin cause conformational changes that alter the affinity of the protein for oxygen. The modulation of oxygen binding allows the O2-transport protein to re￾spond to changes in oxygen demand by tissues. Hemoglobin Subunits Are Structurally Similar to Myoglobin Hemoglobin (Mr 64,500; abbreviated Hb) is roughly spherical, with a diameter of nearly 5.5 nm. It is a tetrameric protein containing four heme prosthetic groups, one associated with each polypeptide chain. Adult hemoglobin contains two types of globin, two
chains (141 residues each) and two  chains (146 residues each). Although fewer than half of the amino acid residues in the polypeptide sequences of the
and  subunits are identical, the three-dimensional struc￾tures of the two types of subunits are very similar. Fur￾thermore, their structures are very similar to that of myoglobin (Fig. 5–6), even though the amino acid se￾quences of the three polypeptides are identical at only 27 positions (Fig. 5–7). All three polypeptides are mem￾bers of the globin family of proteins. The helix-naming convention described for myoglobin is also applied to the hemoglobin polypeptides, except that the
subunit lacks the short D helix. The heme-binding pocket is made up largely of the E and F helices. Heme group Myoglobin b subunit of hemoglobin FIGURE 5–6 A comparison of the structures of myoglobin (PDB ID 1MBO) and the  subunit of hemoglobin (derived from PDB ID 1HGA). L A T V L Mb Hb
Hb Mb Hb
Hb only b Hb V VV E —P LFF — —H A —D EKR L L E —A FLL S ST M—V I LL EPP K —M SSG GAE D7 A G G EHN E DE E1 S S N ACV WKK EAP I LL QTS DQK I LV L NA LVV HVC V V KKK VTV L KT KGA LLL HAA E7 HHH HAA V AL GGG SAH W WW VKK G19 R H H A GG TKK HL F K KK VVV PPG V VV LAL GAK A16 E G — TDG DEE AA— A A FFF D HN LLF H1 G T T V AV GTS APP A GD AND DAP GEE I AG AVV HYV E19 L V L QHQ G GG KAA GAA QAG KHH ASA DEE KVL ML Y I AA GDD NDQ L LL HDN KKK I EG HML AFV R RR EPK LLV L ML ANG EAA F FL EAT LSG KLV F1 L L F FVV B16 S S V KSA RSA C1 H F Y PAT KTN P PP L L DVA ETW ASS I LL T T QDE ATA L KQ SLL H21 A S H ETR F8 HHH KKK HC1 C7 K Y F F9 A A C YYY HC2 F FF T HD KRH HC3 DPE KKK E R HS HL L H26 L F FF KRH G K —G I VV Y HDD G1 P D D Q L LL I PP G KSS KVE D1 T H T YNN 1 1 1 20 20 20 40 40 40 60 60 60 80 80 80 100 100 100 120 120 120 140 140 140 141 146 153 A1 B1 NA1 H and Proximal His Distal His FIGURE 5–7 The amino acid sequences of whale myoglobin and the
and chains of human hemoglobin. Dashed lines mark helix bound￾aries. To align the sequences optimally, short gaps must be introduced into both Hb sequences where a few amino acids are present in the compared sequences. With the exception of the missing D helix in Hb
, this alignment permits the use of the helix lettering convention that emphasizes the common positioning of amino acid residues that are identical in all three structures (shaded). Residues shaded in pink are conserved in all known globins. Note that the common helix-letter￾and-number designation for amino acids does not necessarily corre￾spond to a common position in the linear sequence of amino acids in the polypeptides. For example, the distal His residue is His E7 in all three structures, but corresponds to His64, His58, and His63 in the linear sequences of Mb, Hb
, and Hb, respectively. Nonhelical residues at the amino and carboxyl termini, beyond the first (A) and last (H)
-helical segments, are labeled NA and HC, respectively. 8885d_c05_157-189 8/12/03 8:55 AM Page 163 mac78 mac78:385_REB:

8885dc05_157-1898/12/038:55 AM Page164mac78mac78:385 164 Part I Structure and Catalysis of the ion pairs that stabilize the T' state are broken and some new ones are formed Max Perutz proposed that the T-R transition triggered by changes in the positions of key amino acid side chains surrounding the heme In the T state, the porphyrin is slightly puckered, causing the heme iron to protrude somewhat on the proximal His (His F8) side The binding of O, causes the heme to assume a more planar conformation, shifting the position of the proxi- mal His and the attached F helix (Fig. 5-11). These changes lead to adjustments in the ion pairs at the a,B Hemoglobin Binds Oxygen Cooperatively Hemoglobin must bind oxygen efficiently in the lungs, FIGURE 5-8 Dominant interactions between hemoglobin subunits. where the pO is about 13.3 kPa, and release oxygen in In this representation, a subunits are light and B subunits are dark the tissues, where the pO2 is about 4 kPa. Myoglobin, The strongest subunit interactions(highlighted)occur between unlike or any protein that binds oxygen with a hyperbolic bind subunits. When oxygen binds, the a,B, contact changes little, but ing curve, would be ill-suited to this function, for the there is a large change at the a1B2 contact, with several ion pairs bro. reason illustrated in Figure 5-12. A protein that bound ken(PDB ID 1HGA) Asp FG1 The quaternary structure of hemoglobin features c subunit Lys C5 strong interactions between unlike subunits. The a B, His hc3 interface(and its a,B, counterpart) involves more than 30 residues, and its interaction is sufficiently strong that although mild treatment of hemoglobin with urea tends to cause the tetramer to disassemble into aB dimers, these dimers remain intact. The a B2(and a2B1) inter face involves 19 residues (Fig. 5-8). Hydrophobic in- teractions predominate at the interfaces, but there are also many hydrogen bonds and a few ion pairs(some- times referred to as salt bridges), whose importance is Hemoglobin Undergoes a Structural Change Asp His* NH3 on Binding Oxygen FGI H Arg+. Asp X-ray analysis has revealed two major conformations of hemoglobin: the R state and the T state. Although oxy HC3 H9 gen binds to hemoglobin in either state, it has a signif- NH3 coo icantly higher affinity for hemoglobin in the R state. Oxy- H9 HC3 gen binding stabilizes the R state. When oxygen is Hist As COO absent experimentally, the t state is more stable and is HC3 FG1 thus the predominant conformation of deoxyhemoglo bin. T and R originally denoted"tense"and"relaxed FIGURE 5-9 Some ion pairs that stabilize the T state of deoxyhe- respectively, because the T state is stabilized by a moglobin.(a)A close-up view of a portion of a deoxyhemoglobin greater number of ion pairs, many of which lie at the molecule in the t state(PDB ID 1HGA) Interactions between the ion aiB2(and ceBi interface(Fig. 5-9). The binding of O pairs His HC3 and Asp FGI of the B subunit(blue) and between Ly to a hemoglobin subunit in the T state triggers a change C5 of the a subunit (gray) and His HC3(its a-carboxyl group) of the in conformation to the R state. When the entire protein B subunit hown with dashed lines. (Recall that HC3 is the undergoes this transition, the structures of the individ carboxyl-terminal residue of the B subunit )(b) The interactions be- ual subunits change little, but the aB subunit pairs slide tween these ion pairs, and between others not shown in(a),are past each other and rotate, narrowing the pocket be- schematized in this representation of the extended polypeptide chains tween the B subunits(Fig. 5-10). In this process, some of hemoglobin

of the ion pairs that stabilize the T state are broken and some new ones are formed. Max Perutz proposed that the T n R transition is triggered by changes in the positions of key amino acid side chains surrounding the heme. In the T state, the porphyrin is slightly puckered, causing the heme iron to protrude somewhat on the proximal His (His F8) side. The binding of O2 causes the heme to assume a more planar conformation, shifting the position of the proxi￾mal His and the attached F helix (Fig. 5–11). These changes lead to adjustments in the ion pairs at the
12 interface. Hemoglobin Binds Oxygen Cooperatively Hemoglobin must bind oxygen efficiently in the lungs, where the pO2 is about 13.3 kPa, and release oxygen in the tissues, where the pO2 is about 4 kPa. Myoglobin, or any protein that binds oxygen with a hyperbolic bind￾ing curve, would be ill-suited to this function, for the reason illustrated in Figure 5–12. A protein that bound 164 Part I Structure and Catalysis a1 a2 b1 b2 FIGURE 5–8 Dominant interactions between hemoglobin subunits. In this representation,
subunits are light and  subunits are dark. The strongest subunit interactions (highlighted) occur between unlike subunits. When oxygen binds, the
11 contact changes little, but there is a large change at the
12 contact, with several ion pairs bro￾ken (PDB ID 1HGA). (a) a subunit b subunit Asp FG1 His HC3 Lys C5 COO COO COO Arg+ Lys+ Asp Arg+ Asp Lys+ His+ His+ Asp Asp HC3 FG1 HC3 H9 HC3 FG1 C5 H9 HC3 C5 COO NH3 b2 b1 a2 a1 (b) + NH3 + NH3 + NH3 + FIGURE 5–9 Some ion pairs that stabilize the T state of deoxyhe￾moglobin. (a) A close-up view of a portion of a deoxyhemoglobin molecule in the T state (PDB ID 1HGA). Interactions between the ion pairs His HC3 and Asp FG1 of the  subunit (blue) and between Lys C5 of the
subunit (gray) and His HC3 (its
-carboxyl group) of the  subunit are shown with dashed lines. (Recall that HC3 is the carboxyl-terminal residue of the  subunit.) (b) The interactions be￾tween these ion pairs, and between others not shown in (a), are schematized in this representation of the extended polypeptide chains of hemoglobin. The quaternary structure of hemoglobin features strong interactions between unlike subunits. The
11 interface (and its
22 counterpart) involves more than 30 residues, and its interaction is sufficiently strong that although mild treatment of hemoglobin with urea tends to cause the tetramer to disassemble into
 dimers, these dimers remain intact. The
12 (and
21) inter￾face involves 19 residues (Fig. 5–8). Hydrophobic in￾teractions predominate at the interfaces, but there are also many hydrogen bonds and a few ion pairs (some￾times referred to as salt bridges), whose importance is discussed below. Hemoglobin Undergoes a Structural Change on Binding Oxygen X-ray analysis has revealed two major conformations of hemoglobin: the R state and the T state. Although oxy￾gen binds to hemoglobin in either state, it has a signif￾icantly higher affinity for hemoglobin in the R state. Oxy￾gen binding stabilizes the R state. When oxygen is absent experimentally, the T state is more stable and is thus the predominant conformation of deoxyhemoglo￾bin. T and R originally denoted “tense” and “relaxed,” respectively, because the T state is stabilized by a greater number of ion pairs, many of which lie at the
12 (and
21) interface (Fig. 5–9). The binding of O2 to a hemoglobin subunit in the T state triggers a change in conformation to the R state. When the entire protein undergoes this transition, the structures of the individ￾ual subunits change little, but the
 subunit pairs slide past each other and rotate, narrowing the pocket be￾tween the  subunits (Fig. 5–10). In this process, some 8885d_c05_157-189 8/12/03 8:55 AM Page 164 mac78 mac78:385_REB:

8885ac05157-1898/12/038:55 AM Page165mac78mac78:385p Chapter 5 Protein Function His hc3 AC T state R state FIGURE 5-10 The T-R transition (PDB ID 1HGA and 5-9. The transition from the t state to the r state shifts the subunit hese depictions of deoxyhemoglobin, as in Figure 5-9, the B subunits irs substantially, affecting certain ion pairs. Most noticeab ly, the His re blue and the a subunits are gray. Positively charged side chains HC3 residues at the carboxyl termini of the B subunits, which are and chain termini involved in ion pairs are shown in blue, their neg. volved in ion pairs in the T state, rotate in the r state toward the cen tively charged partners in red. The Lys C5 of each a subunit and Asp ter of the molecule, where they are no longer in ion pairs. Another FG1 of each B subunit are visible but not labeled(compare Fig. 5-9a). dramatic result of the T-R transition is a narrowing of the pocket Note that the molecule is oriented slightly differently than O2 with high affinity would bind it efficiently in the lungs An allosteric protein is one in which the binding but would not release much of it in the tissues. If the of a ligand to one site affects the binding properties of protein bound oxygen with a sufficiently low affinity to another site on the same protein. The term"allosteric release it in the tissues, it would not pick up much oxy- derives from the Greek allos, "other, " and stereos gen in the lungs “ solid”or“ shape." Allosteric proteins are those having Hemoglobin solves the problem by undergoing a" other shapes, " or conformations, induced by the bind transition from a low-affinity state (the T state) to a ing of ligands referred to as modulators. The conforma high-affinity state(the R state)as more Oe molecules tional changes induced by the modulator(s) intercon- are bound. As a result, hemoglobin has a hybrid s- vert more-active and less-active forms of the protein. shaped, or sigmoid, binding curve for oxygen (Fig. The modulators for allosteric proteins may be either 12). A single-subunit protein with a single ligand- inhibitors or activators. When the normal ligand and binding site cannot produce a sigmoid binding curve- even if binding elicits a conformational change- because each molecule of ligand binds independently and cannot affect the binding of another molecule. In Leu Hem contrast, O, binding to individual subunits of hemo- globin can alter the affinity for O2 in adjacent subunits The first molecule of O2 that interacts with deoxyhe- moglobin binds weakly, because it binds to a subunit in the T state. Its binding, however, leads to confor- mational changes that are communicated to adjacent Helix F subunits, making it easier for additional molecules of Leu F4 O to bind. In effect the t-R transition occurs more T state readily in the second subunit once O, is bound to the first subunit. The last (fourth) O2 molecule binds to a FIGURE 5-11 Changes in conformation near heme on O2 binding heme in a subunit that is already in the R state, and to deoxyhemoglobin (Derived from PDB ID 1HGA and 1BBB)The hence it binds with much higher affinity than the first shift in the position of the F helix when heme binds O2 is thought to be one of the adjustments that triggers the T-R transition

O2 with high affinity would bind it efficiently in the lungs but would not release much of it in the tissues. If the protein bound oxygen with a sufficiently low affinity to release it in the tissues, it would not pick up much oxy￾gen in the lungs. Hemoglobin solves the problem by undergoing a transition from a low-affinity state (the T state) to a high-affinity state (the R state) as more O2 molecules are bound. As a result, hemoglobin has a hybrid S￾shaped, or sigmoid, binding curve for oxygen (Fig. 5–12). A single-subunit protein with a single ligand￾binding site cannot produce a sigmoid binding curve— even if binding elicits a conformational change— because each molecule of ligand binds independently and cannot affect the binding of another molecule. In contrast, O2 binding to individual subunits of hemo￾globin can alter the affinity for O2 in adjacent subunits. The first molecule of O2 that interacts with deoxyhe￾moglobin binds weakly, because it binds to a subunit in the T state. Its binding, however, leads to confor￾mational changes that are communicated to adjacent subunits, making it easier for additional molecules of O2 to bind. In effect, the T n R transition occurs more readily in the second subunit once O2 is bound to the first subunit. The last (fourth) O2 molecule binds to a heme in a subunit that is already in the R state, and hence it binds with much higher affinity than the first molecule. An allosteric protein is one in which the binding of a ligand to one site affects the binding properties of another site on the same protein. The term “allosteric” derives from the Greek allos, “other,” and stereos, “solid” or “shape.” Allosteric proteins are those having “other shapes,” or conformations, induced by the bind￾ing of ligands referred to as modulators. The conforma￾tional changes induced by the modulator(s) intercon￾vert more-active and less-active forms of the protein. The modulators for allosteric proteins may be either inhibitors or activators. When the normal ligand and Chapter 5 Protein Function 165 His HC3 His HC3 His HC3 a1 a2 b1 b2 a1 a2 b1 b2 T state R state FIGURE 5–10 The T n R transition. (PDB ID 1HGA and 1BBB) In these depictions of deoxyhemoglobin, as in Figure 5–9, the  subunits are blue and the
subunits are gray. Positively charged side chains and chain termini involved in ion pairs are shown in blue, their neg￾atively charged partners in red. The Lys C5 of each
subunit and Asp FG1 of each  subunit are visible but not labeled (compare Fig. 5–9a). Note that the molecule is oriented slightly differently than in Figure 5–9. The transition from the T state to the R state shifts the subunit pairs substantially, affecting certain ion pairs. Most noticeably, the His HC3 residues at the carboxyl termini of the  subunits, which are in￾volved in ion pairs in the T state, rotate in the R state toward the cen￾ter of the molecule, where they are no longer in ion pairs. Another dramatic result of the T n R transition is a narrowing of the pocket between the  subunits. T state R state Val FG5 Heme O2 Leu FG3 Helix F Leu F4 His F8 FIGURE 5–11 Changes in conformation near heme on O2 binding to deoxyhemoglobin. (Derived from PDB ID 1HGA and 1BBB.) The shift in the position of the F helix when heme binds O2 is thought to be one of the adjustments that triggers the T n R transition. 8885d_c05_157-189 8/12/03 8:55 AM Page 165 mac78 mac78:385_REB:

8885ac05157-1898/12/038:55 AM Page166mac78mac78:385冲g 166 Part I Structure and Catalysis the affinities of any remaining unfilled binding sites, and O, can be considered as both a ligand and an activating homotropic modulator. There is only one binding site High-affinity for O2 on each subunit, so the allosteric effects giving state rise to cooperativity are mediated by conformational = changes transmitted from one subunit to another by subunit-subunit interactions. A sigmoid binding curve is 0.6 diagnostic of cooperative binding. It permits a much more sensitive response to ligand concentration and is important to the function of many multisubunit proteins 0.4 The principle of allostery extends readily to regulatory enzymes, as we shall see in Chapter 6. Cooperative conformational changes depend on 0.2 Low-affinity variations in the structural stability of different parts of a protein, as described in Chapter 4. The binding sites of an allosteric protein typically consist of stable seg ments in proximity to relatively unstable segments, with the latter capable of frequent changes in conformation pO2 (kPa) or disorganized motion(Fig. 5-13). When a ligand binds FIGURE 5-12 A sigmoid(cooperative)binding curve. A sigmoid the moving parts of the proteins binding site may be binding curve can be viewed as a hybrid curve reflecting a transition stabilized in a particular conformation, affecting the from a low-affinity to a high-affinity state. Cooperative binding, as conformation of adjacent polypeptide subunits. If the manifested by a sigmoid binding curve, renders hemoglobin more sensitive to the small differences in O2 concentration between the tis.(a sues and the lungs, allowing hemoglobin to bind oxygen in the lur (where pO, is high) and release it in the tissues(where po, is low). modulator are identical. the interaction is termed ho- motropic. When the modulator is a molecule other than the normal ligand the interaction is heterotropic. Some proteins have two or more modulators and therefore can Binding have both homotropic and heterotropic interactions Cooperative binding of a ligand to a multimeric pro- Ligand tein, such as we observe with the binding of O, to he moglobin, is a form of allosteric binding often observed in multimeric proteins. The binding of one ligand affects FIGURE 5-13 Structural changes in a multisubunit protein under going cooperative binding to ligand. Structural stability is not uniform throughout a protein molecule Shown here is a hypothetical dimeric protein, with regions of high(blue), medium(green), and low (red) stability. The ligand-binding sites are composed of both high- and low- stability segments, so affinity for ligand is relatively low. (a)In the ab- sence of ligand, the red segments are quite flexible and take up a va ty of conformations, few of which facilitate ligand binding. The green segments are most stable in the low-affinity state. (b) The bind ing of ligand to one subunit stabilizes a high-affinity conformation of the nearby red segment(now shown in green), inducing a conforma- tional change in the rest of the polypeptide. This is a form of induced fit. The conformational change is transmitted to the other subunit through protein-protein interactions, such that a higher-affinity con- formation of the binding site is stabilized in the other subunit. (c)A ■ Stable second ligand molecule can now bind to the second subunit, with a higher affinity than the binding of the first, giving rise to the observed Less stable Unstable

modulator are identical, the interaction is termed ho￾motropic. When the modulator is a molecule other than the normal ligand the interaction is heterotropic. Some proteins have two or more modulators and therefore can have both homotropic and heterotropic interactions. Cooperative binding of a ligand to a multimeric pro￾tein, such as we observe with the binding of O2 to he￾moglobin, is a form of allosteric binding often observed in multimeric proteins. The binding of one ligand affects the affinities of any remaining unfilled binding sites, and O2 can be considered as both a ligand and an activating homotropic modulator. There is only one binding site for O2 on each subunit, so the allosteric effects giving rise to cooperativity are mediated by conformational changes transmitted from one subunit to another by subunit-subunit interactions. A sigmoid binding curve is diagnostic of cooperative binding. It permits a much more sensitive response to ligand concentration and is important to the function of many multisubunit proteins. The principle of allostery extends readily to regulatory enzymes, as we shall see in Chapter 6. Cooperative conformational changes depend on variations in the structural stability of different parts of a protein, as described in Chapter 4. The binding sites of an allosteric protein typically consist of stable seg￾ments in proximity to relatively unstable segments, with the latter capable of frequent changes in conformation or disorganized motion (Fig. 5–13). When a ligand binds, the moving parts of the protein’s binding site may be stabilized in a particular conformation, affecting the conformation of adjacent polypeptide subunits. If the 166 Part I Structure and Catalysis FIGURE 5–13 Structural changes in a multisubunit protein under￾going cooperative binding to ligand. Structural stability is not uniform throughout a protein molecule. Shown here is a hypothetical dimeric protein, with regions of high (blue), medium (green), and low (red) stability. The ligand-binding sites are composed of both high- and low￾stability segments, so affinity for ligand is relatively low. (a) In the ab￾sence of ligand, the red segments are quite flexible and take up a va￾riety of conformations, few of which facilitate ligand binding. The green segments are most stable in the low-affinity state. (b) The bind￾ing of ligand to one subunit stabilizes a high-affinity conformation of the nearby red segment (now shown in green), inducing a conforma￾tional change in the rest of the polypeptide. This is a form of induced fit. The conformational change is transmitted to the other subunit through protein-protein interactions, such that a higher-affinity con￾formation of the binding site is stabilized in the other subunit. (c) A second ligand molecule can now bind to the second subunit, with a higher affinity than the binding of the first, giving rise to the observed positive cooperativity. Binding site Binding site Ligand Stable Less stable Unstable (a) (b) (c) 1.0 0.8 0.6 0.2 0.4 0 v 4 8 12 16 pO2 (kPa) pO2 in tissues pO2 in lungs Transition from low- to high￾affinity state Low-affinity state High-affinity state FIGURE 5–12 A sigmoid (cooperative) binding curve. A sigmoid binding curve can be viewed as a hybrid curve reflecting a transition from a low-affinity to a high-affinity state. Cooperative binding, as manifested by a sigmoid binding curve, renders hemoglobin more sensitive to the small differences in O2 concentration between the tis￾sues and the lungs, allowing hemoglobin to bind oxygen in the lungs (where pO2 is high) and release it in the tissues (where pO2 is low). 8885d_c05_157-189 8/12/03 8:55 AM Page 166 mac78 mac78:385_REB: