8885ac05157-1898/12/038:55 AM Page167mac78mac78:385 Chapter 5 Protein Function entire binding site were highly stable, then few struc practice, and the measured value of nH is always less tural changes could occur in this site or be propagated than the actual number of ligand-binding sites in the to other parts of the protein when a ligand binds tein As is the case with myoglobin, ligands other than An H of less than I indicates negative cooperativ oxygen can bind to hemoglobin. An important example in which the binding of one molecule of ligand im- is carbon monoxide, which binds to hemoglobin about pedes the binding of others. Well-documented cases of 250 times better than does oxygen. Human exposure to negative cooperativity are rare CO can have tragic consequences(Box 5-1) To adapt the hill equation to the binding of oxygen hemoglobin we must again substitute pO for L and Cooperative Ligand Binding Can Be Described P5o for Kd Quantitatively n log pO2-n log P5o (5-17) Cooperative binding of oxygen by hemoglobin was first analyzed by Archibald Hill in 1910. From this work came Hill plots for myoglobin and hemoglobin are given in Fig- a general approach to the study of cooperative ligand ure 5-14 binding to multisubunit proteins For a protein with n binding sites, the equilibrium Two Models Suggest Mechani of Equation 5-1 becomes for Cooperative Binding P+nL (5-12) Biochemists now know a great deal about the T and R and the expression for the association constant becomes states of hemoglobin, but much remains to be learned about how the t-R transition occurs Two models for the cooperative binding of ligands to proteins with mul- tiple binding sites have greatly influenced thinking The expression for g(see Egn 5-8)is about this problem The first model was proposed by Jacques Monod 6 Jeffries Wyman, and Jean-Pierre Changeux in 1965, and is called the mwc model or the concerted model (Fig. 5-15a). The concerted model assumes that the Rearranging, then taking the log of both sides, yields subunits of a cooperatively binding protein are func identical, that each subunit can exist in (at 65- where Kd=Lo. Hemoglobin Equation 5-16 is the Hill equation, and a plot of high-affinity log [e/(1-0] versus log L is called a Hill plot. Based on the equation, the Hill plot should have a slope of n. However, the experimentally determined slope actually reflects not the number of binding sites but the degree eD of interaction between them. The slope of a Hill plot is Myoglobin therefore denoted by nH, the Hill coefficient, which is a measure of the degree of cooperativity. If nH equals state 1, ligand binding is not cooperative, a situation that can arise even in a multisubunit protein if the subunits do not communicate. An nH of greater than I indicates positive cooperativity in ligand binding. This is the situation observed in hemoglobin, in which the binding of one molecule of ligand facilitates the binding of FIGURE 5-14 Hill plots for the binding of oxygen to myoglobin and others. The theoretical upper limit for nH is reached hemoglobin. When nH=1, there is no evident cooperativity. The max- when nH=n. In this case the binding would be com- imum degree of cooperativity observed for hemoglobin corresponds pletely cooperative: all binding sites on the protein approximately to nH=3. Note that while this indicates a high level would bind ligand simultaneously, and no protein mol- of cooperativity, nH is less than n, the number of O2-binding sites ecules partially saturated with ligand would be present hemoglobin. This is normal for a protein that exhibits allosteric bind- under any conditions. This limit is never reached in
entire binding site were highly stable, then few structural changes could occur in this site or be propagated to other parts of the protein when a ligand binds. As is the case with myoglobin, ligands other than oxygen can bind to hemoglobin. An important example is carbon monoxide, which binds to hemoglobin about 250 times better than does oxygen. Human exposure to CO can have tragic consequences (Box 5–1). Cooperative Ligand Binding Can Be Described Quantitatively Cooperative binding of oxygen by hemoglobin was first analyzed by Archibald Hill in 1910. From this work came a general approach to the study of cooperative ligand binding to multisubunit proteins. For a protein with n binding sites, the equilibrium of Equation 5–1 becomes P nL PLn (5–12) and the expression for the association constant becomes Ka [ [ P P ] L [L n ] ] n (5–13) The expression for (see Eqn 5–8) is [L] [ n L ] n Kd (5–14) Rearranging, then taking the log of both sides, yields 1 [ K L] d n (5–15) log 1 n log [L] log Kd (5–16) where Kd [L]n 0.5. Equation 5–16 is the Hill equation, and a plot of log [/(1 )] versus log [L] is called a Hill plot. Based on the equation, the Hill plot should have a slope of n. However, the experimentally determined slope actually reflects not the number of binding sites but the degree of interaction between them. The slope of a Hill plot is therefore denoted by nH, the Hill coefficient, which is a measure of the degree of cooperativity. If nH equals 1, ligand binding is not cooperative, a situation that can arise even in a multisubunit protein if the subunits do not communicate. An nH of greater than 1 indicates positive cooperativity in ligand binding. This is the situation observed in hemoglobin, in which the binding of one molecule of ligand facilitates the binding of others. The theoretical upper limit for nH is reached when nH n. In this case the binding would be completely cooperative: all binding sites on the protein would bind ligand simultaneously, and no protein molecules partially saturated with ligand would be present under any conditions. This limit is never reached in yz Chapter 5 Protein Function 167 practice, and the measured value of nH is always less than the actual number of ligand-binding sites in the protein. An nH of less than 1 indicates negative cooperativity, in which the binding of one molecule of ligand impedes the binding of others. Well-documented cases of negative cooperativity are rare. To adapt the Hill equation to the binding of oxygen to hemoglobin we must again substitute pO2 for [L] and P50 n for Kd: log 1 n log pO2 n log P50 n (5–17) Hill plots for myoglobin and hemoglobin are given in Figure 5–14. Two Models Suggest Mechanisms for Cooperative Binding Biochemists now know a great deal about the T and R states of hemoglobin, but much remains to be learned about how the T n R transition occurs. Two models for the cooperative binding of ligands to proteins with multiple binding sites have greatly influenced thinking about this problem. The first model was proposed by Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux in 1965, and is called the MWC model or the concerted model (Fig. 5–15a). The concerted model assumes that the subunits of a cooperatively binding protein are functionally identical, that each subunit can exist in (at 3 log pO2 3 1 1 1 0 3 0 1 2 2 2 2 1 log ) ( Hemoglobin nH 3 Hemoglobin high-affinity state nH 1 Myoglobin nH 1 Hemoglobin low-affinity state nH 1 FIGURE 5–14 Hill plots for the binding of oxygen to myoglobin and hemoglobin. When nH 1, there is no evident cooperativity. The maximum degree of cooperativity observed for hemoglobin corresponds approximately to nH 3. Note that while this indicates a high level of cooperativity, nH is less than n, the number of O2-binding sites in hemoglobin. This is normal for a protein that exhibits allosteric binding behavior. 8885d_c05_157-189 8/12/03 8:55 AM Page 167 mac78 mac78:385_REB:
885c051688/15/0312:03 PM Page168mac78mac78:385REBd 168 Part I Structure and Catalysis BOX 5-1 BIOCHEMISTRY IN MEDICINE Carbon Monoxide: A Stealthy Killer time as people are exposed to a constant low-level Lake Powell, Arizona, August 2000. A family was va source of co cationing in a rented houseboat. They turned on the In an average, healthy in al. 1% or less of the electrical generator to power an air conditioner and a total hemoglobin is complexed as COHb. Since O is television. About 15 minutes later, two brothers, aged a product of tobacco smoke, many smokers have COHb 8 and 11, jumped off the swim deck at the stern. Sit- levels in the range of 3% to 8% of total hemoglobin, uated immediately below the deck was the exhaust and the levels can rise to 15% for chain-smokers port for the generator. Within two minutes, both boys COHb levels equilibrate at 50% in people who breathe were overcome by the carbon monoxide in the ex- air containing 570 ppm of Co for several hours. Rell- haust, which had become able methods have been developed that relate CO con- under the deck. Both drowned. These deaths, along tent in the atmosphere to COHb levels in the blood with a series of deaths in the 1990s linked to house. (Fig. 1). In tests of houseboats with a generator ex- boats of similar design, eventually led to the recall and ust like the one responsible for the Lake Powell redesign of the generator exhaust assembly deaths, CO levels reached 6, 000 to 30,000 ppm under Carbon monoxide(CO), a colorless, odorless gas, the swim deck, and atmospheric O2 levels under the responsible for more than half of yearly deaths due deck declined from 21% to 12%. Even above the swim o poisoning worldwide. CO has an approximately 250- deck, CO levels of up to 7, 200 ppm were detected, fold greater affinity for hemoglobin than does oxygen. high enough to cause death within a few minutes Consequently, relatively low levels of CO can have sub How is a human affected by COHb? At levels of less stantial and tragic effects. When CO combines with than 10% of total hemoglobin, symptoms are rarely ob- hemoglobin, the complex is referred to as carboxyhe- served. At 15%, the individual experiences mild moglobin, or COHb headaches. At 20% to 30%. the headache is severe and Some co is produced by natural processes, but locally high levels generally result only from human activities. Engine and furnace exhausts are important sources, as CO is a byproduct of the incomplete com 12 8 h, light bustion of fossil fuels. In the united states alone xercise nearly 4,000 people succumb to Co poisoning each 10 year, both accidentally and intentionally. Many of the a accidental deaths involve undetected Co buildup in 8 at rest enclosed spaces, such as when a household furnace s8 malfunctions or leaks, venting CO into a home. How ever, Co poisoning can also occur in open spaces, as 1h, light xercise unsuspecting people at work or play inhale the ex haust from generators, outboard motors, tractor en- gines, recreational vehicles, or lawn mowers h at rest Carbon monoxide levels in the atmosphere are rarely dangerous, ranging from less than 0.05 parts per nillion (ppm) in remote and uninhabited areas to 3 to 4 ppm in some cities of the northern hemisphere Carbon monoxide(ppm) In the United States, the government-mandated FIGURE 1 Relationship between the levels of COHb in blood and (Occupational Safety and Health Administration, the concentration of Co in the surrounding air. Four different con- OSHA) limit for CO at worksites is 50 ppm for people ditions of exposure are shown, comparing the effects of short versus working an eight-hour shift. The tight binding of Co extended exposure, and exposure at rest versus exposure during ligh to hemoglobin means that COHb can accumulate over exercise least) two conformations, and that all subunits undergo conformation, but binds each with different affinity Suc- the transition from one conformation to the other si- cessive binding of ligand molecules to the low-affinity multaneously. In this model, no protein has individual conformation(which is more stable in the absence of subunits in different conformations. The two conforma- ligand) makes a transition to the high-affinity confor tions are in equilibrium. The ligand can bind to either mation more likely
168 Part I Structure and Catalysis BOX 5–1 BIOCHEMISTRY IN MEDICINE Carbon Monoxide: A Stealthy Killer Lake Powell, Arizona, August 2000. A family was vacationing in a rented houseboat. They turned on the electrical generator to power an air conditioner and a television. About 15 minutes later, two brothers, aged 8 and 11, jumped off the swim deck at the stern. Situated immediately below the deck was the exhaust port for the generator. Within two minutes, both boys were overcome by the carbon monoxide in the exhaust, which had become concentrated in the space under the deck. Both drowned. These deaths, along with a series of deaths in the 1990s linked to houseboats of similar design, eventually led to the recall and redesign of the generator exhaust assembly. Carbon monoxide (CO), a colorless, odorless gas, is responsible for more than half of yearly deaths due to poisoning worldwide. CO has an approximately 250- fold greater affinity for hemoglobin than does oxygen. Consequently, relatively low levels of CO can have substantial and tragic effects. When CO combines with hemoglobin, the complex is referred to as carboxyhemoglobin, or COHb. Some CO is produced by natural processes, but locally high levels generally result only from human activities. Engine and furnace exhausts are important sources, as CO is a byproduct of the incomplete combustion of fossil fuels. In the United States alone, nearly 4,000 people succumb to CO poisoning each year, both accidentally and intentionally. Many of the accidental deaths involve undetected CO buildup in enclosed spaces, such as when a household furnace malfunctions or leaks, venting CO into a home. However, CO poisoning can also occur in open spaces, as unsuspecting people at work or play inhale the exhaust from generators, outboard motors, tractor engines, recreational vehicles, or lawn mowers. Carbon monoxide levels in the atmosphere are rarely dangerous, ranging from less than 0.05 parts per million (ppm) in remote and uninhabited areas to 3 to 4 ppm in some cities of the northern hemisphere. In the United States, the government-mandated (Occupational Safety and Health Administration, OSHA) limit for CO at worksites is 50 ppm for people working an eight-hour shift. The tight binding of CO to hemoglobin means that COHb can accumulate over time as people are exposed to a constant low-level source of CO. In an average, healthy individual, 1% or less of the total hemoglobin is complexed as COHb. Since CO is a product of tobacco smoke, many smokers have COHb levels in the range of 3% to 8% of total hemoglobin, and the levels can rise to 15% for chain-smokers. COHb levels equilibrate at 50% in people who breathe air containing 570 ppm of CO for several hours. Reliable methods have been developed that relate CO content in the atmosphere to COHb levels in the blood (Fig. 1). In tests of houseboats with a generator exhaust like the one responsible for the Lake Powell deaths, CO levels reached 6,000 to 30,000 ppm under the swim deck, and atmospheric O2 levels under the deck declined from 21% to 12%. Even above the swim deck, CO levels of up to 7,200 ppm were detected, high enough to cause death within a few minutes. How is a human affected by COHb? At levels of less than 10% of total hemoglobin, symptoms are rarely observed. At 15%, the individual experiences mild headaches. At 20% to 30%, the headache is severe and FIGURE 1 Relationship between the levels of COHb in blood and the concentration of CO in the surrounding air. Four different conditions of exposure are shown, comparing the effects of short versus extended exposure, and exposure at rest versus exposure during light exercise. 14 100 8 h, light exercise 1 h, light exercise 8 h, at rest 1 h, at rest 12 COHb in blood (%)10 8 6 4 2 0 0 20 40 Carbon monoxide (ppm) 60 80 least) two conformations, and that all subunits undergo the transition from one conformation to the other simultaneously. In this model, no protein has individual subunits in different conformations. The two conformations are in equilibrium. The ligand can bind to either conformation, but binds each with different affinity. Successive binding of ligand molecules to the low-affinity conformation (which is more stable in the absence of ligand) makes a transition to the high-affinity conformation more likely. 8885d_c05_168 8/15/03 12:03 PM Page 168 mac78 mac78:385_REB: