CH 9: Hemoglobin, Ligand Binding, and Allostery
Modulators of Allosteric Enzymes
Allosteric regulators disrupt the R<->T equilibrium when they bind the enzyme. Inhibitors stabilize the T state while activators stabilize the R state. The disruption of the T<->R equilibrium by substrates is called the homotropic effect. The disruption of the T<->R equilibrium by regulators is called the heterotrophic effect.
Allosterically regulated enzymes do not conform to...
Allosterically regulated enzymes do not conform to Michaelis-menten kinetics • The reaction velocity of allosteric enzymes displays a sigmoidal relationship to substrate concentration.
Allostery vs. cooperative binding
Allostery refers to the coupling of conformational changes between two widely separated sites in a protein. Cooperative binding is a special case of allostery. A macromolecule exhibits cooperative binding if its affinity for its ligand changes with the amount of ligand already bound. If the binding becomes stronger there is positive cooperativity. If the binding becomes weaker there is negative cooperativity as other ligand binding sites become occupied.
High altitude adaptations
At 3000m (10,000 ft) 70% O2 Increase BPG in erythrocytes 4mM to 8mM
Receptor ligand bonding
Binding: R + L ⇌ RL Unbinding: RL ⇌ R+L Kd = [R][L]/[RL] No intermediates! RL is the product her, not the intermediate. This is how hemoglobin binding is different. Bound oxygen hemoglobin complex is the product.
Y=?
Define fraction of receptor (R) bound to a ligand (L) as Y: Y=[L] / Kd+[L]
Fetal hemoglobin
Fetal hemoglobin must bind oxygen when the mother's hemoglobin is releasing oxygen. In fetal hemoglobin, the B subunit is replaced with a γ subunit. The fetal α₂γ₂ hemoglobin does not bind 2,3- BPG as well as adult hemoglobin. The reduced affinity for 2,3-BPG results in fetal hemoglobin having a higher affinity for oxygen, binding oxygen when the mother's hemoglobin is releasing oxygen.
Functional magnetic resonance fMRI can...
Functional magnetic resonance fMRI can distinguish between oxygen and deoxy hemoglobin • The magnetic properties of the heme iron change when it moves into the plane of the protoporphyrin ring. • Functional magnetic resonance can be used to monitor activity in specific regions of the brain by measuring the increase in oxyhemoglobin.
Hemoglobin exists in two distinct states...
Hemoglobin exists in two distinct states: 1. T state - The "Tense" state - Has less affinity for oxygen and is called deoxyhemoglobin. 2. R state - The "Relaxed" state - Has high affinity for oxygen and is called oxyhemoglobin *Of note: In the concerted mode of cooperativity, the hemoglobin must either be in its T state or R state. In the sequential mode of cooperativity, the conformation state of the monomer changes as it binds to oxygen. Hemoglobin response does not fit completely to either model.
Hemoglobin vs. Myoglobin
Hemoglobin is a red blood cell protein that carries oxygen from the lungs to the tissues Myoglobin binds oxygen in muscle cells Hemoglobin is an allosteric protein that displays cooperatively in oxygen binding and release. The binding of oxygen by myoglobin is not cooperative.
Receptor-ligand binding: single binding site What is the shape and equation of the graph?
Hyperbolic y=x / b+x Y axis: fraction bound X axis: [L] *remember, Kd is the value of [L] in which 1/2 of receptors are bound
The Hill coefficient
It describes the fraction of the macromolecule saturated by ligand as a function of the ligand concentration it is used in determining the degree of cooperativeness of the ligand binding to the enzyme or receptor. Positive cooperativity: n > 1 Negative cooperativity: n < 1 Non-cooperative: n = 1 Theoretical maximum cooperativity = # of binding sites
Kd is...
Kd is an expression for how TIGHT a ligand binds to a protein. Dissociation constant Kd = [R][L]/[RL] Kd units: concentration
Chemical basis of the Bohr Effect
Low pH allows the formation of ionic interactions that stabilize the T state of hemoglobin, enhancing oxygen release HbO₂ + H⁺ ⇌ HbH⁺ + O₂
Quantitative measure of Myoglobin-O2 binding Define units and relevant equations
Mb + O2 ⇌ MbO2 Kd Kd = [Mb][O2] / [MbO2] The ligand concentrations, [O2], is given as pO2, or the partial pressure of oxygen. The units are torr. The fractional saturation, Yo₂, is given by the following: [O2] / Kd+[O2] *note, same as: Y = [L] / Kd+[L] Kd = p₅₀, Kd gives the partial pressure of oxygen required to give a fractional saturation of Y=0.5. In the case of myoglobin, the KD is 2-3 torr.
Myoglobin binding curve explained
Myoglobin (Mb) Monomeric (tertiary structure) Contains a single heme group with a bound Fe2+ Binds 1 oxygen molecule per molecule of protein.. Carries O2 from capillaries to sites of usage in cells. (i.e. mitrochondria) Non-cooperative binding of O2. Hyperbolic
Pure hemoglobin vs. Hemoglobin with allosteric regulator oxygen binding curve
Pure hemoglobin without 2,3-BPG does not have a stabilized T state and thus has a higher oxygen affinity and a lower Kd/tight ligand bond (Notice that the black curve is farther to the left and steeper/more hyperbolic). Notice how at 20 torr there is approx. 85% fractional saturation of hemoglobin (resembles behavior of myoglobin). Hemoglobin (as it behave in red blood cells) with 2,3-BPG has a stabilized T state which allows it to behave cooperatively and release oxygen as pO2 decreases. Thus has a higher Kd and less tight ligand bond (notice that the pink curve is farther to the right and less steep/more sigmoidal). At 20 torr there is only about 30% fractional saturation vs approx 100% at 100 torr. 2,3-BPG stabilizes T state of hemoglobin allowing it to be an effective oxygen transporter from the lungs to peripheral tissues.
an allosteric regulator changes the oxygen affinity of hemoglobin
- 2,3-Bisphosphoglycerate (2,3-BPG) present in red blood cells, stabilizes the T state of hemoglobin and thus facilitates the release of oxygen. - 2,3-BPG binds to a pocket in the hemoglobin tetramer that exists only when hemoglobin is in the T state.
Hemoglobin structure
- Tetrametric, two alpha subunits and two beta subunits (Quaternary Structure) - The quaternary structure is best described as a pair of identical alpha-beta dimers (a₁B₁ and a₂B₂) - Each subunit contains a bound heme-Fe2+ - each subunit is structurally similar to myoglobin - Binds a total of 4 oxygen molecules to its four heme groups. - Carries O2 from lungs to tissues, increasing the solubility of O2 in blood - Positive cooperativity in binding of O2; the binding affinity increases as more O2 are bound. **General features of oxygen transport: Oxygen is absolutely required for life in most organisms. All tissues need oxygen. Oxygen is usually taken up in the lungs by the protein hemoglobin and carried throughout the body in the circulatory system. In some cases, there is a need to store large quantities of oxygen in the tissue itself. In this case a specialized oxygen storage protein, myoglobin, is used to store the oxygen and to facilitate its diffusion within cells.
Myoglobin oxygen binding
- single polypeptide chain consisting mainly of a-helices arranged to form a globular structure. Helices are labeled A-H -Heme prosthetic group (porphyrin) binds oxygen via Fe2+ (ferrous form) -iron can form two additional bonds, called the fifth and sixth coordination sites - Monomeric (tertiary structure) - Contains a single heme group with a bound Fe2+ - Binds 1 oxygen molecule per molecule of protein.. - Carries O2 from capillaries to sites of usage in cells. (i.e. mitrochondria) - Non-cooperative binding of O2. General features of oxygen transport: Oxygen is absolutely required for life in most organisms. All tissues need oxygen. Oxygen is usually taken up in the lungs by the proteinhemoglobin and carried throughout the body in the circulatory system. In some cases, there is a need to store large quantities of oxygen in the tissue itself. In this case a specialized oxygen storage protein, myoglobin, is used to store the oxygen and to facilitate its diffusion within cells.
Conformational changes upon O2 binding
1. The ferrous (Fe2+) iron ion lies in the middle of the protoporphyrin bound to 4 nitrogens. 2. Iron can form two additional bonds, called the fifth and sixth coordination sites. 3. The fifth coordination site is occupied by an imidazole ring of a histidine called the proximal histidine. The sixth coordination site binds oxygen. 4. Upon oxygen binding, the iron moves into the plane of the protoporphryin ring.
2,3-BPG favors the....
2,3-BPG favors the T state
distal histidine
A histidine in globins that forms a hydrogen bond to the bound oxygen that helps to prevent the release of superoxide anion. A histidine located near the heme group in myoglobin and hemoglobin that helps maintain the heme iron in the Fe2+ oxidation state and inhibits carbon monoxide binding. It is on the opposite side of the heme from the proximal histidine.
Ligand
A molecule that binds specifically to a protein target (receptor site) of another molecule.
Hill plot
A plot of log (Y/1-Y) vs. log L is called a hill plot. Where n is the Hill coefficient (unique for each system) Turns sigmoid into straight lines. Slope = n (# of binding sites). Allows measurement of binding sites that are cooperative. -Shows that Hb changes from T to R state occurs as more oxygen molecules bound -As some oxygen is bound, additional oxygen bound more avidly -In lungs, when [O2] is high, Hb saturated with 4 O2 as it changes into R state -In tissues, when [O2] is low, Hb rapidly loses 4 oxygen as it goes back to T state
Receptor-ligand binding: multiple binding site What is the shape of the graph?
A sigmoidal binding curve is indicative of a system exhibiting cooperative ligand bonding. It can be viewed as a hybrid curve reflecting a transition from low affinity to high affinity state. Y axis: fraction bound X axis: [L] binding of a ligand at one site can affect the binding at other sites multiple binding sites can lead to sigmoidal binding curves
The glucocorticoid receptor is a protein that plays a key role in the body's inflammation response. The drug hydrocortisone binds this protein with a Kd=20nM, and the drug dexamethasone binds it with a Kd = 2 nM. Which of the following statements is true? A. Dexamethasone has a lower Kd and binds with a higher affinity B. Hydrocortisone has a higher Kd and binds with higher affinity C. The glucocorticoid receptor will have Y=0.5 when [hydrocortisone] = [dexamethasone] D. more information is needed to say anything about binding
A. Dexamethasone has a lower Kd and binds with a higher affinity B. Hydrocortisone has a higher Kd and binds with higher affinity C. The glucocorticoid receptor will have Y=0.5 when [hydrocortisone] = [dexamethasone] D. more information is needed to say anything about binding
Enzyme 1 Enzyme 2 Consider the hypothetical metabolic pathway: A ----------- > B --------- > C occurring at 30°C. The conversion of A to B has a positive ΔG°; yet there is net conversion of A to B during the normal functioning of the pathway in cells. Explain how this is likely to occur.
The conversion of A to B can be made spontaneous (i.e. ΔG<0) if the concentration of B relative to that of A is maintained at a low level (lower than the ratio of B to A at equilibrium). The reaction will proceed forward as the system moves towards equilibrium. This is an example of indirect coupling.
How does cooperativity enhance oxygen delivery by hemoglobin?
The cooperativeness of hemoglobin makes it a much more efficient transporter of oxygen than myoglobin. Myoglobin and hemoglobin both become highly saturated with oxygen at high concentrations (E.g. ~90% saturated at 100 torr in the lungs) Hemoglobin is characterized by much weaker binding to oxygen at low concentrations compared to myoglobin (At 20 torr in the tissues compare 30% hemoglobin saturation to 85% myoglobin saturation). The cooperativeness of tetramers work both ways in hemoglobin. As one oxygen molecule binds to one heme group, the oxygen affinity for the other groups increase. Once an oxygen molecule is released, this stimulates the release of the other oxygen molecules. This makes hemoglobin ideal in transporting and releasing oxygen from lungs to tissues where it is needed. Myoglobin is not cooperative and binds strongly to oxygen even at low partial pressures, making it unsuited for oxygen transport from lungs to tissues.
Bohr Effect
The tendency of certain factors to stabilize the hemoglobin in the T state, thus reducing its affinity for oxygen and enhancing the release of oxygen to the tissues. The factors include increased PCO2 and decreased pH. Note that the Bohr effect shifts the oxy-hemolobin saturation curve to the right. Describes the pH dependence of Hb's affinity for O2 -at decreased pH, Hb has a lower affinity for oxygen Carbon dioxide and H+, produced by actively respiring tissues, enhance oxygen release by hemoglobin. The stimulation of oxygen release by H+ is called the Bohr effect.
Transition from T to R state (deoxy to oxy)
The transition from deoxyhemoglobin (T state) to oxyhemoglobin (R state) occurs upon oxygen binding. When oxygen binds, the proximal histidine moves with the iron, thus the α-helix that the proximal His is in also moves. The resulting structural change is communicated to the other subunits so that the two αβ dimers rotate with respect to one another, resulting in the formation of the R state. helpful link: http://biochem.web.utah.edu/iwasa/projects/hemoglobin.html
proximal histidine
This amino acid residue is sterically repelled by the porphyrin ring and pulls the iron away from the heme plane. O2 binding pulls the iron back in it's spot. The residue occupying the fifth coordination site to which iron can bind in hemoglobin and myoglobin
What is Y when [L]=Kd?
Y (fraction of receptor bound to a ligand) is 0.5 when [L]=Kd
Receptor
a protein the binds a ligand
noncooperative binding
a situation in which binding of a ligand to a macromolecule does not affect the affinities of other binding sites on the same molecule
Receptor-ligand binding: multiple binding site
binding of a ligand at one site can affect the binding at other sites multiple binding sites can lead to sigmoidal binding curves
negative cooperativity
first binding event reduces affinity at remaining sites a cooperative effect whereby binding of the first ligand to an enzyme or protein causes the affinity for the next ligand to be lower
positive cooperativity
the first substrate changes the shape of the enzyme allowing other substrates to bind more easily first binding event increases affinity at remaining sites recognized by sigmoidal binding curves
Allosteric Enzymes Depend on Alterations in Quaternary Structure... explain.
• All allosteric enzymes display quaternary structure with multiple active sites and regulatory sites. • Two models are used to explain the behavior of allosteric enzymes
The threshold effect
• Allosteric enzymes are more sensitive to changes in substrate concentration near their Km values than are Michaelis-Menten enzymes. • This sensitivity is called the threshold effect.
Hydrogen Ions and Carbon Dioxide Promote the Release of Oxygen
• Carbon dioxide is transported to the lungs as bicarbonate. • Carbonic anhydrase facilitates the formation of bicarbonate ions.
H+ and CO2 are heterotrophic regulators
• Carbon dioxide reacts with terminal amino groups of the a-chains to form negatively charged carbamate groups. The carbamate forms salt bridges that stabilize the T state. • Carbon dioxide and H+ are heterotropic regulators of oxygen binding by hemoglobin. Oxygen is a homotropic regulator of its own binding.
