Allosteric Effects & Cooperative Binding

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1 Allosteric Effects & Cooperative Binding The shape of the binding curve for oxygen to myoglobin is hyperbolic and follows the equation for non-cooperative binding: Y=[L]/(KD + [L]). The binding curve for oxygen to hemoglobin is sigmoidal in shape, showing a relatively low oxygen affinity at low oxygen concentrations to facilitate the unloading of oxygen in the tissues. At higher oxygen concentrations the affinity of hemoglobin for oxygen increases, such that the hemoglobin becomes fully saturated with oxygen in the lungs. This mode of binding is an example of positive cooperative binding that is caused by the fact that oxygen binding to one subunit of hemoglobin causes the other subunits to change and adopt a different, or allosteric structure. The oxygen induced structural changes in hemoglobin are illustrated in the Jmol on the right. After starting the animation you should see that the relative orientation of the four subunits in hemoglobin change as the protein goes from the low affinity T state to the high affinity R state. Allosteric effects are widely found in biological systems and play an important role in the regulation of many biological processes in addition to oxygen transport. A large number of enzymes that are involved in metabolism are under allosteric control. Allosteric effects allow a biological system to quickly fine-tune its properties in response to changes in the intracellular environment. Cooperative/Allosteric behavior requires the presence of: 1. At least two binding sites. 2. Two forms of the macromolecule. One form, usually called the T or tense state, binds the primary ligand (e.g. oxygen) with low affinity. The other form, usually called the R or relaxed state, binds ligand with high affinity. The T and R states are in equilibrium with each other. In the case of positive cooperativity the fraction of T states exceeds that of the R state and the binding of ligand increases the amount of the R state, thus increases the ease of ligand binding.

2 In the case of negative cooperativity, the fraction of the T state is smaller than that of the R state. Thus, the initial binding affinity is high. However, the binding of ligand increases the amount of the T state, thus reducing the binding affinity. Heterotropic and Homotropic Allosteric Moderators: Homotropic: If the two ligands are the same (e.g. oxygen affecting its own binding) then this is called a homotropic allosteric effect. Example shown below is a positive homotropic modulator; it increases the affinity of the system. An example of a homotropic allosteric activator. In this example, a homodimeric protein contains a single binding site on each monomer. In the absence of bound ligand the protein is largely in the T state and shows low affinity for the ligand. The protein is in equilibrium with the R-state and a sub-population can be found in the R-form. Ligand binding to the R-form stabilizes that conformation and increases the likelihood that the other binding site will exist in the R-form. The second ligand binds with high affinity to the pre-existing R-state. Heterotropic If the two ligands are different and affect each others binding, it is called a heterotropic allosteric effect. The following example shows both a heterotropic negative allosteric modulator (I), and a positive allosteric activator (A). Heterotropic inhibitors and activators. In this example the protein contains three different binding sites, one for the ligand, one for the activator, and one for the inhibitor. The inhibitor binds to the T-state

3 with high affinity and the activator binds to the R-state with high affinity. The ligand (blue square) binds to the R-form with high affinity. As the inhibitor concentration increases the amount of protein in the R-form decreases, reducing the affinity for the ligand. As the activator concentration increases, the amount of the R- form increases, and so does the ligand binding affinity. Allosteric effects in Hemoglobin Homotropic activation by Oxygen. The binding of oxygen to one of the four subunits of hemoglobin causes the remaining subunits to prefer the R-state, thus increasing the affinity of subsequent binding events. The other units sense the binding of one oxygen by the following mechanism: Binding of O2 to Fe 2+ in heme moves the proximal His residue and its attached helix (helix-f) Helix F adjusts its conformation by movement of the α and β subunits. Change in interaction between the α and β subunits causes a conformational change to the R-state. Heterotropic Allosteric Effectors in Hemoglobin. There are many heterotropic allosteric effectors of oxygen binding in hemoglobin. Two examples are: 1. Protons: Oxygen affinity is decreased at low ph, such as in active muscle. This drop in ph is due to the production of carbonic acid from the CO2 (released during aerobic metabolism) and lactic acid (released during anaerobic metabolism). This provides an immediate response to the metabolic state of the tissue. The lower ph causes more oxygen to be released. This effect is called the Bohr effect, after the scientist who initially characterized it. 2. bis-phosphoglycerate (BPG). This compound binds to the deoxy (tense) form of hemoglobin, therefore it reduces oxygen affinity. The levels of BPG are increased at high altitudes to increase the amount of oxygen that is delivered to the tissue. This is an adaptive response, requiring several days at high altitude. The production of excess BPG, although it reduces the oxygen affinity, makes hemoglobin more efficient at delivering oxygen to the tissues. The molecular nature of the action of BPG is quite clear: In deoxy hemoglobin, a positively charged binding pocket exists between two of the four subunits. Thus BPG can easily bind, and

4 when it does so, it stabilizes the deoxy, or tense, form of the protein. In oxy-hemoglobin, the relative movement of the chains that occurs during the allosteric transition to the R state closes this pocket, so BPG can no longer fit. BPG binding site on deoxyhemoglobin (T form). BPG binding site on oxy-hemoglobin (R-form).

5 Example - Adaptive Effect of BPG on O2 Delivery This graph shows the effect of BPG (bisphosphoglycerate) on the oxygen affinity of normal hemoglobin. The level of BPG in the blood at sea level is ~5mM. After adaptation to high altitudes in 2-4 days the BPG level rises to about 8 mm. Since BPG binds to the T-state, the binding affinity of O2 to Hb is reduced as the BPG concentration increases. [Note, this curve is somewhat different than previous lecture notes because it includes the effect of blood ph and salt on the oxygen affinity. Also note [O2] is in kpa.] Question: Use this chart to calculate the oxygen delivery for unadapted and adapted hemoglobin in the Rocky mountains ([O2] = 8 kpa), assuming that the oxygen concentration in the tissues is 4 kpa. Compare this to the amount of oxygen delivered at sea level for unadapted hemoglobin. Answer: The amount of oxygen bound to hemoglobin will be proportional to the fractional saturation, Y, which can be read from the curve. Conditions [O2] Y, 5 mm BPG. Y, 8 mm BPG. Rockies 8 kpa Tissue 4 kpa Δ Oxygen delivery at sea level, for 5 mm BPG, is = 0.40, or 40%. Prior to adaptation, oxygen delivery in the Rockies is reduced to 34%. After the levels of BPG are increased to 8mM, the oxygen delivery is increased to 40%.