Special Focus 473 transmitted to the subunit interfaces, where they trigger conformational readjust- ments that lead to the rupture of interchain salt links. The Oxy and Deoxy Forms of Hemoglobin Represent Two Different Conformational States Hemoglobin resists oxygenation (see Figure 15.20) because the deoxy form is sta- bilized by specific hydrogen bonds and salt bridges (ion-pair bonds) (Figure 15.27). All of these interactions are broken in oxyhemoglobin, as the molecule sta- bilizes into a new conformation. The shift in helix F upon oxygenation leads to rupture of the Tyr 145:Val 98 hydrogen bond. In deoxyhemoglobin, with these interactions intact, the C-termini of the four subunits are restrained, and this con- formational state is termed T, the tense or taut form. In oxyhemoglobin, these C-termini have almost complete freedom of rotation, and the molecule is now in its R, or relaxed, form. The Allosteric Behavior of Hemoglobin Has Both Symmetry (MWC) Model and Sequential (KNF) Model Components Oxygen is accessible only to the heme groups of the ␣-chains when hemoglobin is in the T conformational state. Max Perutz has pointed out that the heme environ- ment of -chains in the T state is virtually inaccessible because of steric hindrance by amino acid residues in the E helix. This hindrance disappears when the hemo- globin molecule undergoes transition to the R conformational state. Binding of O 2 to the -chains is thus dependent on a T-to-R conformational shift, and this shift is triggered by the subtle changes that occur when O 2 binds to the ␣-chain heme groups. Together these observations lead to a model that is partially MWC and par- tially KNF: O 2 binding to one ␣-subunit and then the other leads to sequential changes in conformation, followed by a switch in quaternary structure at the HbϺ2O 2 state from T to R. Thus, the real behavior of this protein is an amalgam of the two prominent theoretical models for allosteric behavior. H ϩ Promotes the Dissociation of Oxygen from Hemoglobin Protons, carbon dioxide, and chloride ions, as well as the metabolite 2,3- bisphosphoglycerate (or BPG), all affect the binding of O 2 by hemoglobin. Their ef- fects have interesting ramifications, which we shall see as we discuss them in turn. De- oxyhemoglobin has a higher affinity for protons than oxyhemoglobin. Thus, as the pH decreases, dissociation of O 2 from hemoglobin is enhanced. In simple symbolism, ignoring the stoichiometry of O 2 or H ϩ involved: HbO 2 ϩ H ϩ 34HbH ϩ ϩ O 2 (a) (b) FIGURE 15.27 Salt bridges between different subunits in human deoxyhemoglobin.These noncovalent, electro- static interactions are disrupted upon oxygenation. (a) A focus on those salt bridges and hydrogen bonds involving interactions between N-terminal and C-terminal residues in the ␣-chains. Residues in the lower center are Arg ␣ 1 141 (green) with Val ␣ 1 1 (purple), Asp ␣ 2 126 (orange), Lys ␣ 2 127 (yellow), and Val  2 34 (olive); residues at top are Val ␣ 1 93 (yellow) with Tyr ␣ 1 140 (purple). (b) A focus on those salt bridges and hydrogen bonds involving C-terminal residues of -chains:Val  2 78 (olive) with Tyr  2 145 (purple); His  2 146 (light blue) with Asp  2 94 (orange) and Lys ␣ 1 40 (yellow) (pdb id ϭ 2HHB). A DEEPER LOOK Changes in the Heme Iron upon O 2 Binding In deoxyhemoglobin, the six d electrons of the heme Fe 2ϩ exist as four unpaired electrons and one electron pair, and five ligands can be accommodated: the four N-atoms of the porphyrin ring sys- tem and histidine F8. In this electronic configuration, the iron atom is paramagnetic and in the high-spin state. When the heme binds O 2 as a sixth ligand, these electrons are rearranged into three e Ϫ pairs and the iron changes to the low-spin state and is diamagnetic. This change in spin state allows the bond between the Fe 2ϩ ion and histidine F8 to become perpendicular to the heme plane and to shorten. In addition, interactions between the porphyrin N atoms and the iron strengthen. Also, high-spin Fe 2ϩ has a greater atomic volume than low-spin Fe 2ϩ because its four unpaired e Ϫ occupy four orbitals rather than two when the elec- trons are paired in low-spin Fe 2ϩ . So, low-spin iron is less sterically hindered and able to move nearer to the porphyrin plane. 474 Chapter 15 Enzyme Regulation Expressed another way, H ϩ is an antagonist of oxygen binding by Hb, and the satu- ration curve of Hb for O 2 is displaced to the right as acidity increases (Figure 15.28). This phenomenon is called the Bohr effect, after its discoverer, the Danish physiol- ogist Christian Bohr (the father of Niels Bohr, the atomic physicist). The effect has important physiological significance because actively metabolizing tissues produce acid, promoting O 2 release where it is most needed. About two protons are taken up by deoxyhemoglobin. The N-termini of the two ␣-chains and the His 146 residues have been implicated as the major players in the Bohr effect. (The pK a of a free amino terminus in a protein is about 8.0, but the pK a of a protein histidine imidazole is around 6.5.) Neighboring carboxylate groups of Asp 94 residues help stabilize the protonated state of the His 146 imidazoles that occur in deoxyhemo- globin. However, when Hb binds O 2 , changes in the conformation of -chains upon Hb oxygenation move the negative Asp function away, and dissociation of the imidazole protons is favored. CO 2 Also Promotes the Dissociation of O 2 from Hemoglobin Carbon dioxide has an effect on O 2 binding by Hb that is similar to that of H ϩ , partly because it produces H ϩ when it dissolves in the blood: The enzyme carbonic anhydrase promotes the hydration of CO 2 . Many of the protons formed upon ionization of carbonic acid are picked up by Hb as O 2 dissociates. The bicarbonate ions are transported with the blood back to the lungs. When Hb be- comes oxygenated again in the lungs, H ϩ is released and reacts with HCO 3 Ϫ to re- form H 2 CO 3 , from which CO 2 is liberated. The CO 2 is then exhaled as a gas. In addition, some CO 2 is directly transported by hemoglobin in the form of car- bamate (ONHCOO Ϫ ). Free ␣-amino groups of Hb react with CO 2 reversibly: This reaction is driven to the right in tissues by the high CO 2 concentration; the equilibrium shifts the other way in the lungs where [CO 2 ] is low. Thus, carbamyla- tion of the N-termini converts them to anionic functions, which then form salt links with the cationic side chains of Arg ␣141 that stabilize the deoxy or T state of hemoglobin. ϩRO CO 2 NH 2 ϩROONH H ϩ COO Ϫ CO 2 H 2 OH 2 CO 3 HCO 3 Ϫ H ϩ ϩϩ carbonic anhydrase carbonic acid bicarbonate 100 80 60 40 20 0 Percent saturation 0 20 40 60 80 100 120 140 pO 2 , torr Myoglobin Arterial pO 2 Venous pO 2 pH 7.6 pH 7.4 pH 7.2 pH 7.0 pH 6.8 FIGURE 15.28 The oxygen saturation curves for myoglo- bin and for hemoglobin at five different pH values: 7.6, 7.4, 7.2, 7.0, and 6.8. Special Focus 475 In addition to CO 2 , Cl Ϫ and BPG also bind better to deoxyhemoglobin than to oxyhemoglobin, causing a shift in equilibrium in favor of O 2 release. These various effects are demonstrated by the shift in the oxygen saturation curves for Hb in the presence of one or more of these substances (Figure 15.29). Note that the O 2 - binding curve for Hb ϩ BPG ϩ CO 2 fits that of whole blood very well. 2,3-Bisphosphoglycerate Is an Important Allosteric Effector for Hemoglobin The binding of 2,3-bisphosphoglycerate (BPG) to Hb promotes the release of O 2 (Figure 15.29). Erythrocytes (red blood cells) normally contain about 4.5 mM BPG, a concentration equivalent to that of tetrameric hemoglobin molecules. Interestingly, this equivalence is maintained in the HbϺBPG binding stoichiome- try because the tetrameric Hb molecule has but one binding site for BPG. This site is situated within the central cavity formed by the association of the four sub- units. The strongly negative BPG molecule (Figure 15.30) is electrostatically bound via interactions with the positively charged functional groups of each Lys 82, His 2, His 143, and the NH 3 ϩ -terminal group of each -chain. These positively charged residues are arranged to form an electrostatic pocket complementary to the conformation and charge distribution of BPG (Figure 15.31). In effect, BPG crosslinks the two -subunits. The ionic bonds between BPG and the two -chains aid in stabilizing the conformation of Hb in its deoxy form, thereby favoring the dissociation of oxygen. In oxyhemoglobin, this central cavity is too small for BPG to fit. Or, to put it another way, the conformational changes in the Hb molecule that accompany O 2 binding perturb the BPG-binding site so that BPG can no longer be accommodated. Thus, BPG and O 2 are mutually exclusive allosteric ef- fectors for Hb, even though their binding sites are physically distinct. BPG Binding to Hb Has Important Physiological Significance The importance of the BPG effect is evident in Figure 15.29. Hemoglobin stripped of BPG is virtually saturated with O 2 at a pO 2 of only 20 torr, and it cannot release its oxygen within tissues, where the pO 2 is typically 40 torr. BPG shifts the oxygen satu- ration curve of Hb to the right, making the Hb an O 2 delivery system eminently suited to the needs of the organism. BPG serves this vital function in humans, most primates, and a number of other mammals. However, the hemoglobins of cattle, sheep, goats, deer, and other animals have an intrinsically lower affinity for O 2 , and these Hbs are relatively unaffected by BPG. Fetal Hemoglobin Has a Higher Affinity for O 2 Because It Has a Lower Affinity for BPG The fetus depends on its mother for an adequate supply of oxygen, but its circulatory system is entirely independent. Gas exchange takes place across the placenta. Ideally 20 0 20 40 60 pO 2 , torr 100 80 60 40 Percent O 2 saturation Stripped Hb Hb + CO 2 Hb + BPG Hb + BPG + CO 2 Whole blood FIGURE 15.29 Oxygen-binding curves of blood and of hemoglobin in the absence and presence of CO 2 and BPG. From left to right: stripped Hb, Hb ϩ CO 2 , Hb ϩ BPG, Hb ϩ BPG ϩ CO 2 , and whole blood. HC OPO 3 2 – C PO H 2 C OPO 3 2 – OO – O – O – O C H H C H C O O O P O – O – O – FIGURE 15.30 The structure, in ionic form, of BPG or 2,3-bisphosphoglycerate, an important allosteric effector for hemoglobin. 476 Chapter 15 Enzyme Regulation then, fetal Hb should be able to absorb O 2 better than maternal Hb so that an effective transfer of oxygen can occur. Fetal Hb differs from adult Hb in that the -chains are replaced by very similar, but not identical, 146-residue subunits called ␥-chains (gamma chains). Fetal Hb is thus ␣ 2 ␥ 2 . Recall that BPG functions through its interaction with the -chains. BPG binds less effectively with the ␥-chains of fetal Hb (also called Hb F). (Fetal ␥-chains have Ser instead of His at position 143 and thus lack two of the positive charges in the central BPG-binding cavity.) Figure 15.32 compares the relative affinities of adult Hb (also known as Hb A) and Hb F for O 2 under similar conditions of pH and [BPG]. Note that Hb F binds O 2 at pO 2 values where most of the oxygen has dissoci- ated from Hb A. Much of the difference can be attributed to the diminished capacity of Hb F to bind BPG (compare Figures 15.29 and 15.32); Hb F thus has an intrinsically greater affinity for O 2 , and oxygen transfer from mother to fetus is ensured. Sickle-Cell Anemia Is Characterized by Abnormal Red Blood Cells In 1904, a Chicago physician treated a 20-year-old black college student complaining of headache, weakness, and dizziness. The blood of this patient revealed serious anemia—only half the normal number of red cells were present. Many of these cells 100 80 60 40 20 Percent O 2 saturation 20 40 60 80 100 pO 2 , torr Hb F Hb A FIGURE 15.32 Comparison of the oxygen saturation curves of Hb A and Hb F under similar conditions of pH and [BPG]. FIGURE 15.31 The ionic binding of BPG to the two -subunits of Hb. BPG lies at center of the cavity be- tween the two -subunits. The highlighted residues are N-terminal Val  1 and Val  2 (yellow), His  1 2, His  2 2, His  1 143, and His  2 143 (purple), Lys  1 82 and Lys  2 82 (green) (pdb id ϭ 1B86). Special Focus 477 were abnormally shaped; in fact, instead of the characteristic disc shape, these ery- throcytes were elongated and crescentlike in form, a feature that eventually gave name to the disease sickle-cell anemia. These sickle cells pass less freely through the capillaries, impairing circulation and causing tissue damage. Furthermore, these cells are more fragile and rupture more easily than normal red cells, leading to anemia. Sickle-Cell Anemia Is a Molecular Disease A single amino acid substitution in the -chains of Hb causes sickle-cell anemia. Re- placement of the glutamate residue at position 6 in the -chain by a valine residue marks the only chemical difference between Hb A and sickle-cell hemoglobin, Hb S. The amino acid residues at position 6 lie at the surface of the hemoglobin molecule. In Hb A, the ionic R groups of the Glu residues fit this environment. In contrast, the aliphatic side chains of the Val residues in Hb S create hydrophobic protrusions where none existed before. To the detriment of individuals who carry this trait, a hy- drophobic pocket forms in the EF corner of each -chain of Hb when it is in the de- oxy state, and this pocket nicely accommodates the Val side chain of a neighboring Hb S molecule (Figure 15.33). This interaction leads to the aggregation of Hb S mol- ecules into long, chainlike polymeric structures. The obvious consequence is that de- oxyHb S is less soluble than deoxyHb A. The concentration of hemoglobin in red blood cells is high (about 150 mg/mL), so even in normal circumstances it is on the HUMAN BIOCHEMISTRY Hemoglobin and Nitric Oxide Nitric oxide (NO и) is a simple gaseous molecule whose many re- markable physiological functions are still being discovered. For ex- ample, NO и is known to act as a neurotransmitter and as a second messenger in signal transduction (see Chapter 32). Furthermore, endothelial relaxing factor (ERF, also known as endothelium- derived relaxing factor, or EDRF), an elusive hormonelike agent that acts to relax the musculature of the walls (endothelium) of blood vessels and lower blood pressure, has been identified as NO и. It has long been known that NO и is a high-affinity ligand for Hb, binding to its heme-Fe 2ϩ atom with an affinity 10,000 times greater than that of O 2 . An enigma thus arises: Why isn’t NO и in- stantaneously bound by Hb within human erythrocytes and pre- vented from exerting its vasodilation properties? The reason that Hb doesn’t block the action of NO и is due to a unique interaction between Cys 93 of Hb and NO и discovered by Li Jia, Celia and Joseph Bonaventura, and Johnathan Stamler at Duke University. Nitric oxide reacts with the sulfhydryl group of Cys 93, forming an S-nitroso derivative: This S-nitroso group is in equilibrium with other S-nitroso com- pounds formed by reaction of NO и with small-molecule thiols such as free cysteine or glutathione (an isoglutamylcysteinylgly- cine tripeptide): OOCH 2 NOSO These small-molecule thiols serve to transfer NO и from erythro- cytes to endothelial receptors, where it acts to relax vascular ten- sion. NO и itself is a reactive free-radical compound whose biolog- ical half-life is very short (1–5 sec). S-nitrosoglutathione has a half-life of several hours. The reactions between Hb and NO и are complex. NO и forms a ligand with the heme-Fe 2ϩ that is quite stable in the absence of O 2 . However, in the presence of O 2 , NO и is oxidized to NO 3 Ϫ and the heme-Fe 2ϩ of Hb is oxidized to Fe 3ϩ , forming methemoglobin. Fortunately, the interaction of Hb with NO и is controlled by the allosteric transition between R-state Hb (oxyHb) and T-state Hb (deoxyHb). Cys 93 is more exposed and reactive in R-state Hb than in T-state Hb, and binding of NO и to Cys 93 precludes re- action of NO и with heme iron. Upon release of O 2 from Hb in tis- sues, Hb shifts conformation from R state to T state, and binding of NO и at Cys 93 is no longer favored. Consequently, NO и is re- leased from Cys 93 and transferred to small-molecule thiols for delivery to endothelial receptors, causing capillary vasodilation. This mechanism also explains the puzzling observation that free Hb produced by recombinant DNA methodology for use as a whole-blood substitute causes a transient rise of 10 to 12 mm Hg in diastolic blood pressure in experimental clinical trials. (Con- ventional whole-blood transfusion has no such effect.) It is now ap- parent that the “synthetic” Hb, which has no bound NO и, is bind- ing NO и in the blood and preventing its vasoregulatory function. In the course of hemoglobin evolution, the only invariant amino acid residues in globin chains are His F8 (the obligatory heme ligand) and a Phe residue acting to wedge the heme into its pocket. However, in mammals and birds, Cys 93 is also invariant, no doubt due to its vital role in NO и delivery. Adapted from Jia, L., et al., 1996. S-Nitrosohaemoglobin: A dynamic activ- ity of blood involved in vascular control. Nature 380:221–226. OOCH 2 OCH 2 OC OSONO CCH 2 O COO Ϫ COO Ϫ H 3 ϩ N H C H C OO N H N H OO OO O CH 2 S-nitroso g lutathione 478 Chapter 15 Enzyme Regulation verge of crystallization. The formation of insoluble deoxyHb S fibers distorts the red cell into the elongated sickle shape characteristic of the disease. 2 2 In certain regions of Africa, the sickle-cell trait is found in 20% of the people. Why does such a dele- terious heritable condition persist in the population? For reasons as yet unknown, individuals with this trait are less susceptible to the most virulent form of malaria. The geographic distribution of malaria and the sickle-cell trait are positively correlated. Oxy- hemoglobin A (a) (b) β 1 β 2 α 1 α 2 β 1 β 2 α 1 α 2 β 1 β 2 α 1 α 2 β 1 β 2 α 1 α 2 Deoxy- hemoglobin A Oxy- hemoglobin S Deoxy- hemoglobin S α 1 α 2 β 1 β 2 Deoxyhemoglobin S polymerizes into filaments α 1 α 2 β 1 β 2 α 1 α 2 β 1 β 2 β 1 β 2 α 1 α 2 β 1 β 2 α 1 α 2 β 1 β 2 α 1 α 2 (c) (d) ANIMATED FIGURE 15.33 The polymerization of Hb S via the interactions between the hydrophobic Val side chains at position 6 and the hydrophobic pockets in the EF corners of -chains in neigh- boring Hb molecules. (a) The protruding “block”on Oxy S represents the Val hydrophobic protrusion.The com- plementary hydrophobic pocket in the EF corner of deoxy -chains is represented by a square-shaped indenta- tion. (This indentation is probably present in Hb A also.) Only the  2 Val protrusions and the  1 EF pockets are shown. (The  1 Val protrusions and the  2 EF pockets are not involved, although they are present.) (b) The polymerization of Hb S via  2 Val6 insertions into neighboring  1 pockets. (c) Molecular graphic of an Hb S dimer of tetramers.  2 Val residues are highlighted in blue; heme is shown in red (pdb id ϭ 2HBS). (d) Molecular graphic of the Hb S filament (pdb id ϭ 2HBS). See this figure animated at www.cengage.com/login. SUMMARY 15.1 What Factors Influence Enzymatic Activity? The two prominent ways to regulate enzyme activity are (1) to increase or decrease the num- ber of enzyme molecules or (2) to increase or decrease the intrinsic activity of each enzyme molecule. Changes in enzyme amounts are typ- ically regulated via gene expression and protein degradation. Changes in the intrinsic activity of enzyme molecules are achieved principally by allosteric regulation or covalent modification. 15.2 What Are the General Features of Allosteric Regulation? Allosteric enzymes show a sigmoid response of velocity, v, to increasing [S], indicating that binding of S to the enzyme is cooperative. Allosteric enzymes often are susceptible to feedback inhibition. Allosteric enzymes may also respond to allosteric activation. Allosteric activators signal a need for the end product of the pathway in which the allosteric enzyme functions. As a general rule, allosteric enzymes are oligomeric, with each monomer possessing a substrate-binding site and an allosteric site where effectors bind. Interaction of one subunit of an allosteric enzyme with its substrate (or its effectors) is communicated to the other subunits of the enzyme through intersubunit interactions. These interactions can lead to conformational transitions that make it easier (or harder) for addi- tional equivalents of ligand (S, A, or I) to bind to the enzyme. 15.3 Can Allosteric Regulation Be Explained by Conformational Changes in Proteins? Monod, Wyman, and Changeux postulated that the subunits of allosteric enzymes can exist in two conformational states (R and T), that all subunits in any enzyme molecule are in the same conformational state (symmetry), that equilibrium strongly favors the T conformational state, and that S binds preferentially (“only”) to the R state. Sigmoid binding curves result, provided that [T 0 ] ϾϾ [R 0 ] in the absence of S and that S binds “only” to R. Positive or negative effectors influence the relative T/R equilibrium by binding preferen- tially to T (negative effectors) or R (positive effectors), and the sub- strate saturation curve is shifted to the rig ht (negative effectors) or left (positive effectors). Problems 479 In an alternative allosteric model suggested by Koshland, Nemethy, and Filmer (the KNF model), S binding leads to conformational changes in the enzyme. The altered conformation of the enzyme may display higher affinity for the substrate (positive cooperativity) or lower affinity for the substrate or other ligand (negative cooperativity). Negative co- operativity is not possible within the MWC model. Reversible changes in the oligomeric state of a protein can also yield allosteric behavior. For ex- ample, a monomer–oligomer equilibrium for an allosteric protein, where only the oligomer binds S and [monomer] ϾϾ [oligomer], would show cooperative substrate binding. 15.4 What Kinds of Covalent Modification Regulate the Activity of Enzymes? Reversible phosphorylation is the most prominent form of covalent modification in cellular regulation. Phosphorylation is accom- plished by protein kinases; phosphoprotein phosphatases act in the reverse direction to remove the phosphate group. Regulation must be imposed on these converter enzymes so that their enzyme targets adopt the metabolically appropriate state (active versus inactive). Thus, these converter enzymes are themselves the targets of allosteric regulation or covalent modification. Although several hundred chemical modifica- tions of proteins have been described, only a few are used for reversible conversion of enzymes between active and inactive forms. Besides phos- phorylation, these regulatory types include adenylylation, uridylylation, ADP-ribosylation, methylation, and oxidation-reduction of protein disulfide bonds. 15.5 Is the Activity of Some Enzymes Controlled by Both Allosteric Regulation and Covalent Modification? Some enzymes are subject to both allosteric regulation and regulation by covalent modification. A prime example is glycogen phosphorylase. Glycogen phosphorylase exists in two forms, a and b, which differ only in whether or not Ser 14 - OH is phosphorylated (a) or not (b). Glycogen phosphorylase b shows positive cooperativity in binding its substrate, phosphate. In addition, glycogen phosphorylase b is allosterically activated by the positive effec- tor AMP. In contrast, ATP and glucose-6-P are negative effectors for glycogen phosphorylase b. Covalent modification of glycogen phospho- rylase b by phosphorylase kinase converts it from a less active, allosteri- cally regulated form to the more active a form that is less responsive to allosteric regulation. Glycogen phosphorylase is both activated and freed from allosteric control by covalent modification. Special Focus: Is There an Example in Nature That Exemplifies the Relationship Between Quaternary Structure and the Emergence of Al- losteric Properties? Hemoglobin and Myoglobin—Paradigms of Pro- tein Structure and Function Myoglobin and hemoglobin have illumi- nated our understanding of protein structure and function. Myoglobin is monomeric, whereas hemoglobin has a quaternary structure. Myo- globin functions as an oxygen-storage protein in muscle; Hb is an O 2 - transport protein. When Mb binds O 2 , its heme iron atom is drawn within the plane of the heme, slightly shifting the position of the F helix of the protein. Hemoglobin shows cooperative binding of O 2 and allo- steric regulation by H ϩ , CO 2 , and 2,3-bisphosphoglycerate. The allosteric properties of Hb can be traced to the movement of the F helix upon O 2 binding to Hb heme groups and the effects of F-helix movement on in- teractions between the protein’s subunits that alter the intrinsic affinity of the other subunits for O 2 . The allosteric transitions in Hb partially conform to the MWC model in that a concerted conformational change from a T-state, low-affinity conformation to an R-state, high-affinity form takes place after 2 O 2 are bound (by the 2 Hb ␣-subunits). However, Hb also behaves somewhat according to the KNF model of allostery in that oxygen binding leads to sequential changes in the conformation and O 2 affinity of hemoglobin subunits. Sickle-cell anemia is a molecular disease traceable to a tendency for Hb S to polymerize as a consequence of hav- ing a E6V amino acid substitution that creates a “sticky” hydrophobic patch on the Hb surface. PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. List six general ways in which enzyme activity is controlled. 2. Why do you suppose proteolytic enzymes are often synthesized as inactive zymogens? 3. (Integrates with Chapter 13.) Draw both Lineweaver–Burk plots and Hanes–Woolf plots for an MWC allosteric enzyme system, show- ing separate curves for the kinetic response in (a) the absence of any effectors, (b) the presence of allosteric activator A, and (c) the presence of allosteric inhibitor I. 4. The KNF model for allosteric transitions includes the possibility of negative cooperativity. Draw Lineweaver–Burk and Hanes–Woolf plots for the case of negative cooperativity in substrate binding. (As a point of reference, include a line showing the classic Michaelis– Menten response of v to [S].) 5. The equation ϭ n allows the calculation of Y (the fractional saturation of hemoglobin with O 2 ), given P 50 and n (see box on page 472). Let P 50 ϭ 26 torr and n ϭ 2.8. Calculate Y in the lungs, where pO 2 ϭ 100 torr, and Y in the capillaries, where pO 2 ϭ 40 torr. What is the efficiency of O 2 delivery under these condi- tions (expressed as Y lungs Ϫ Y capillaries )? Repeat the calculations, but for n ϭ 1. Compare the values for Y lungs Ϫ Y capillaries for n ϭ 2.8 versus Y lungs Ϫ Y capillaries for n ϭ 1 to determine the effect of coop- erative O 2 binding on oxygen delivery by hemoglobin. 6. The cAMP formed by adenylyl cyclase (Figure 15.18) does not persist because 5Ј-phosphodiesterase activity prevalent in cells hydrolyzes cAMP to give 5Ј-AMP. Caffeine inhibits 5Ј-phosphodiesterase activity. Describe the effects on glycogen phosphorylase activity that arise as a consequence of drinking lots of caffeinated coffee. pO 2 ᎏ P 50 Y ᎏ (1 Ϫ Y) 7. If no precautions are taken, blood that has been stored for some time becomes depleted in 2,3-BPG. What happens if such blood is used in a transfusion? 8. Enzymes have evolved such that their K m values (or K 0.5 values) for substrate(s) are roughly equal to the in vivo concentration(s) of the substrate(s). Assume that glycogen phosphorylase is assayed at [P i ] Ϸ K 0.5 in the absence and presence of AMP or ATP. Estimate from Figure 15.14 the relative glycog en phosphorylase activity when (a) neither AMP or ATP is present, (b) AMP is present, and (c) ATP is present. (Hint: Use a ruler to get relative values for the velocity v at the appropriate midpoints of the saturation curves.) 9. Cholera toxin is an enzyme that covalently modifies the G ␣ -subunit of G proteins. (Cholera toxin catalyzes the transfer of ADP-ribose from NAD ϩ to an arginine residue in G ␣ , an ADP-ribosylation reac- tion.) Covalent modification of G ␣ inactivates its GTPase activity. Predict the consequences of cholera toxin on cellular cAMP and glycogen levels. 10. Allosteric enzymes that sit at branch points leading to several essen- tial products sometimes display negative cooperativity for feedback inhibition (allosteric inhibition) by one of the products. What might be the advantage of negative cooperativity instead of positive cooperativity in feedback inhibitor binding by such enzymes? 11. Consult Table 15.2 and a. Suggest a consensus amino acid sequence within phosphorylase kinase that makes it a target of protein kinase A (the cAMP- dependent protein kinase). b. Suggest an effective amino acid sequence for a regulatory domain pseudosubstrate sequence that would exert intrasteric control on phosphorylase kinase by blocking its active site. 12. What are the relative advantages (and disadvantages) of allosteric regulation versus covalent modification? 480 Chapter 15 Enzyme Regulation 13. You land a post as scientific investigator with a pharmaceutical com- pany that would like to develop drugs to treat people with sickle-cell anemia. They want ideas from you! What molecular properties of Hb S might you suggest as potential targets of drug therapy? 14. Under appropriate conditions, nitric oxide (NO и) combines with Cys 93 in hemoglobin and influences its interaction with O 2 . Is this interaction an example of allosteric regulation or covalent modification? 15. Lactate, a metabolite produced under anaerobic conditions in mus- cle, lowers the affinity of myoglobin for O 2 . This effect is beneficial, because O 2 dissociation from Mb under anaerobic conditions will provide the muscle with oxygen. Lactate binds to Mb at a site dis- tinct from the O 2 -binding site at the heme. In light of this observa- tion, discuss whether myoglobin should be considered an allosteric protein. 16. An allosteric model based on multiple oligomeric states of a pro- tein has been proposed by E. K. Jaffe (2005. Morpheeins: A new structural paradigm for allosteric regulation. Trends in Biochemical Sciences 30:490–497). This model coins the term morpheeins to de- scribe the different forms of a protein that can assume more than one conformation, where each distinct conformation assembles into an oligomeric structure with a fixed number of subunits. For example, conformation A of the protein monomer forms trimers, whereas conformation B of the monomer forms tetramers. If trimers and tetramers have different kinetic properties (K m and k cat values), as in low-activity trimers and high-activity tetramers, then the morpheein ensemble behaves like an allosterically regulated enzyme. Drawing on the traditional MWC model as an analogy, di- agram a simple morpheein model in which wedge-shaped protein monomers assemble into trimers but the alternative conformation for the monomer (a square shape) forms tetramers. Further, the substrate, S, or allosteric regulator, A, binds “only” to the square conformation, and its binding prevents the square from adopting the wedge conformation. Describe how your diagram yields allo- steric behavior. 17. CTP synthetase catalyzes the synthesis of CTP from UTP: UTP ϩ ATP ϩ glutamine st CTP ϩ glutamate ϩ ADP ϩ P i The substrates UTP and ATP show positive cooperativity in their binding to the enzyme, which is an ␣ 4 -type homotetramer. However, the other substrate, glutamine, shows negative cooperativity. Draw substrate saturation curves of the form v versus [S]/K 0.5 for each of these three substrates that illustrate these effects. 18. Glyceraldehyde-3-phosphate dehydrogenase catalyzes the synthesis of 1,3-bisphosphoglycerate: Glyceraldehyde-3-P ϩ P i ϩ NAD ϩ st 1,3-BPG ϩ NADH ϩ H ϩ The enzyme is a tetramer. NAD ϩ binding shows negative coopera- tivity. Draw a diagram of possible conformational states for this tetrameric enzyme and its response to NAD ϩ binding that illustrates negative cooperativity. Preparing for the MCAT Exam 19. On the basis of the graphs shown in Figures 15.28 and 15.29 and the relationship between blood pH and respiration (Chapter 2), predict the effect of hyperventilation and hypoventilation on Hb:O 2 affinity. 20. Figure 15.17 traces the activation of glycogen phosphorylase from hormone to phosphorylation of the b form of glycogen phosphory- lase to the a form. These effects are reversible when hormone dis- appears. Suggest reactions by which such reversibility is achieved. FURTHER READING General References Fersht, A., 1999. Structure and Mechanism in Protein Science: A Guide to En- zyme Catalysis and Protein Folding. New York: W. H. Freeman. Protein Kinases Johnson, L., 2007. Protein kinases and their therapeutic exploitation. Transactions 35:7–11. Manning, G., et al., 2002. The protein kinase complement of the human genome. Science 298:1912–1934. A catalog of the protein kinase genes identified within the human genome. About 2% of all eukary- otic genes encode protein kinases. Allosteric Regulation Changeux, J P., and Edelstein, S. J., 2005. Allosteric mechanisms of sig- nal transduction. Science 308:1424–1428. Helmstaedt, K., Krappman, S., and Braus, G. H., 2001. Allosteric regula- tion of catalytic activity. Escherichia coli aspartate transcarbamoylase versus yeast chorismate mutase. Microbiology and Molecular Biology Re- views 65:404–421. The authors present evidence to show that the MWC two-state model is oversimplified, as Monod, Wyman, and Changeux themselves originally stipulated. Koshland, D. E., Jr., and Hamadani, K., 2002. Proteomics and models for enzyme cooperativity. Journal of Biological Chemistry 277:46841–46844. An overview of both the MWC and the KNF models for allostery and a discussion of the relative merits of these models. The fact that the number of allosteric enzymes showing negative cooperativity is about the same as the number showing positive cooperativity is an impor- tant focus of this review. Koshland, D. E., Jr., Nemethy, G., and Filmer, D., 1966. Comparison of experimental binding data and theoretical models in proteins con- taining subunits. Biochemistry 5:365–385. The KNF model. Kuriyan, J., and Eisenberg, D., 2007. The origin of protein interactions and allostery in colocalization. Nature 450:983–990. Monod, J., Wyman, J., and Changeux, J-P., 1965. On the nature of allo- steric transitions: A plausible model. Journal of Molecular Biology 12: 88–118. The classic paper that provided the first theoretical analysis of allosteric regulation. Schachman, H. K., 1990. Can a simple model account for the allosteric transition of aspartate transcarbamoylase? Journal of Biological Chem- istry 263:18583–18586. Tests of the postulates of the allosteric mod- els through experiments on aspartate transcarbamoylase. Swain, J. F., and Gierasch, L. M., 2006. The changing landscape of pro- tein allostery. Current Opinion in Structural Biology 16:102–108. Glycogen Phosphorylase Johnson, L. N., and Barford, D., 1993. The effects of phosphorylation on the structure and function of proteins. Annual Review of Biophysics and Biomolecular Structure 22:199–232. Johnson, L. N., and Barford, D., 1994. Electrostatic effects in the con- trol of glycogen phosphorylase by phosphorylation. Protein Science 3:1726–1730. Lin, K., et al., 1996. Comparison of the activation triggers in yeast and muscle glycogen phosphorylase. Science 273:1539–1541. Lin, K., et al., 1997. Distinct phosphorylation signals converge at the cat- alytic center in glycogen phosphorylases. Structure 5:1511–1523. Rath, V. L., et al., 1996. The evolution of an allosteric site in phosphory- lase. Structure 4:463–473. Hemoglobin Ackers, G. K., 1998. Deciphering the molecular code of hemoglobin al- lostery. Advances in Protein Chemistry 51:185–253. Dickerson, R. E., and Geis, I., 1983. Hemoglobin: Structure, Function, Evo- lution and Pathology. Menlo Park, CA: Benjamin/Cummings. Henr y, E. R., et al., 2002. A tertiary two-state allosteric model for hemo- globin. Biophysical Chemistry 98:149–164. Weiss, J. N., 1997. The Hill equation revisited: Uses and abuses. The FASEB Journal 11:835–841. © Bettmann/CORBIS 16 Molecular Motors 16.1 What Is a Molecular Motor? Motor proteins, also known as molecular motors, use chemical energy (ATP) to or- chestrate movements, transforming ATP energy into the mechanical energy of mo- tion. In all cases, ATP hydrolysis is presumed to drive and control protein confor- mational changes that result in sliding or walking movements of one molecule relative to another. To carry out directed movements, molecular motors must be able to associate and dissociate reversibly with a polymeric protein array, a surface or sub- structure in the cell. ATP hydrolysis drives the process by which the motor protein ratchets along the protein array or surface. As fundamental and straightforward as all this sounds, elucidation of these basically simple processes has been extremely challenging for biochemists, involving the application of many sophisticated chemi- cal and physical methods in many different laboratories. This chapter describes the structures and chemical functions of molecular motor proteins and some of the ex- periments by which we have come to understand them. Molecular motors may be linear or rotating. Linear motors crawl or creep along a polymer lattice, whereas rotating motors consist of a rotating element (the “rotor”) and a stationary element (the “stator”), in a fashion much like a simple electrical motor. The linear motors we will discuss include kinesins and dyneins (which crawl along microtubules), myosin (which slides along actin filaments in muscle), and DNA helicases (which move along a DNA lattice, unwinding duplex DNA to form single-stranded DNA). Rotating motors include the flagellar motor complex, de- scribed in this chapter, and the ATP synthase, which will be described in Chapter 20. 16.2 What Is the Molecular Mechanism of Muscle Contraction? Muscle Contraction Is Triggered by Ca 2؉ Release from Intracellular Stores Muscle contraction is the result of interactions between myosin and actin, the two predominant muscle proteins. Thick filaments of myosin slide along thin fila- ments of actin to cause contraction. The cells of skeletal muscle are long and multinucleate and are referred to as muscle fibers. Skeletal muscles in higher animals consist of 100-m-diameter fibers, some as long as the muscle itself. Each of these muscle fibers contains hundreds of myofibrils (Figure 16.1), each of which spans the length of the fiber and is about Michelangelo’s David epitomizes the musculature of the human form. Buying bread from a man in Brussels He was six foot four and full of muscles I said “Do you speak-a my language?” He just smiled and gave me a Vegemite sandwich. Colin Hay and Ron Strykert, lyrics from Down Under KEY QUESTIONS 16.1 What Is a Molecular Motor? 16.2 What Is the Molecular Mechanism of Muscle Contraction? 16.3 What Are the Molecular Motors That Orchestrate the Mechanochemistry of Microtubules? 16.4 How Do Molecular Motors Unwind DNA? 16.5 How Do Bacterial Flagella Use a Proton Gradient to Drive Rotation? ESSENTIAL QUESTION Movement is an intrinsic property associated with all living things.Within cells, mole- cules undergo coordinated and organized movements, and cells themselves may move across a surface. At the tissue level, muscle contraction allows higher organisms to carry out and control crucial internal functions, such as peristalsis in the gut and the beating of the heart. Muscle contraction also enables the organism to perform orga- nized and sophisticated movements, such as walking, running, flying,and swimming. How can biological macromolecules, carrying out conformational changes on the molecular level, achieve these feats of movement that span the microscopic and macroscopic worlds? Create your own study path for this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login. 482 Chapter 16 Molecular Motors Sarcolemma Sarcoplasmic reticulum Terminal cisternae Transverse tubule Nucleus Contractile filaments Myofibril Mitochondrion SR membrane Transverse tubule FIGURE 16.1 The structure of a skeletal muscle cell, showing the manner in which transverse tubules en- able the sarcolemmal membrane to extend into the in- terior of the fiber.T-tubules and sarcoplasmic reticulum (SR) membranes are juxtaposed at structures termed triad junctions (inset). HUMAN BIOCHEMISTRY Smooth Muscle Effectors Are Useful Drugs Not all vertebrate muscle is skeletal muscle. Vertebrate organisms employ smooth muscle for long, slow, and involuntary contractions in various organs, including large blood vessels, intestinal walls, the gums of the mouth, and in the female, the uterus. Smooth muscle contraction is triggered by Ca 2ϩ -activated phosphorylation of myosin by myosin light-chain kinase (MLCK). The action of epi- nephrine and related agents forms the basis of therapeutic control of smooth muscle contraction. Breathing disorders, including asthma and various allergies, can result from excessive contraction of bronchial smooth muscle tissue. Treatment with epinephrine, whether by tablets or aerosol inhalation, inhibits MLCK and relaxes bronchial muscle tissue. More specific bronchodilators, such as albuterol (see accompanying figure), act more selectively on the lungs and avoid the undesirable side effects of epinephrine on the heart. Albuterol is also used to prevent premature labor in pregnant women because of its relaxing effect on uterine smooth muscle. Conversely, oxytocin, known also as Pitocin, stimulates contraction of uterine smooth muscle. This natural secretion of the pituitary gland is often administered to induce labor. CH 2 OH HO CH 2 CH 3 CCN OH H H CH 3 CH 3 H 3 N GlnProLeuGly Cys Asn Ile CysTyr COO Ϫ ϩ S S Albuterol Oxytocin (Pitocin) ᮡ The structure of oxytocin.