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 accompanied 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 hindrance. When O2 binds to free heme, the axis of the oxygen 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 hybrid 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 binding is physiologically important, because CO is a lowlevel byproduct of cellular metabolism. Other factors, not yet well-defined, also seem to modulate the interaction of heme with CO in these proteins. The binding of O2 to the heme in myoglobin also depends on molecular motions, or “breathing,” in the protein 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 ligandbinding site. If the protein were rigid, O2 could not enter or leave the heme pocket at a measurable rate. However, rapid molecular flexing of the amino acid side chains produces transient cavities in the protein structure, and O2 evidently makes its way in and out by moving through these cavities. Computer simulations of rapid structural fluctuations in myoglobin suggest that there are many such pathways. One major route is provided 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 critical 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 daughter cells that form large amounts of hemoglobin and then lose their intracellular organelles—nucleus, mitochondria, 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 dissolved 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) Carbon monoxide binds to free heme with the CO axis perpendicular to the plane of the porphyrin ring. When binding to the heme in myoglobin, CO is forced to adopt a slight angle because the perpendicular arrangement is sterically blocked by His E7, the distal His. This effect 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 hydrogen-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. Hemoglobin, with its multiple subunits and O2-binding sites, is better suited to oxygen transport. As we shall see, interactions 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 respond 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 structures of the two types of subunits are very similar. Furthermore, their structures are very similar to that of myoglobin (Fig. 5–6), even though the amino acid sequences of the three polypeptides are identical at only 27 positions (Fig. 5–7). All three polypeptides are members 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 boundaries. 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-letterand-number designation for amino acids does not necessarily correspond 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 proximal 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 binding 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 broken (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 deoxyhemoglobin. (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 between 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) interface involves 19 residues (Fig. 5–8). Hydrophobic interactions predominate at the interfaces, but there are also many hydrogen bonds and a few ion pairs (sometimes 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 oxygen binds to hemoglobin in either state, it has a significantly higher affinity for hemoglobin in the R state. Oxygen binding stabilizes the R state. When oxygen is absent experimentally, the T state is more stable and is thus the predominant conformation of deoxyhemoglobin. 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 individual subunits change little, but the subunit pairs slide past each other and rotate, narrowing the pocket between 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 oxygen 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 Sshaped, or sigmoid, binding curve for oxygen (Fig. 5–12). A single-subunit protein with a single ligandbinding 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 hemoglobin can alter the affinity for O2 in adjacent subunits. The first molecule of O2 that interacts with deoxyhemoglobin binds weakly, because it binds to a subunit in the T state. Its binding, however, leads to conformational 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 binding of ligands referred to as modulators. The conformational changes induced by the modulator(s) interconvert 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 negatively 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 involved in ion pairs in the T state, rotate in the R state toward the center 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 homotropic. 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 protein, such as we observe with the binding of O2 to hemoglobin, 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 segments 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 undergoing 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 lowstability segments, so affinity for ligand is relatively low. (a) In the absence of ligand, the red segments are quite flexible and take up a variety of conformations, few of which facilitate ligand binding. The green segments are most stable in the low-affinity state. (b) The binding of ligand to one subunit stabilizes a high-affinity conformation of the nearby red segment (now shown in green), inducing a conformational 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 conformation 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 highaffinity 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 tissues 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: