Biochemistry I, Fall Term Lecture 13 Sept 28, 20051Lecture 13: Allosteric Effects and Cooperative BindingAssigned reading in Campbell: Chapter 4.7, 7.2Key Terms:• Homotropic Allosteric effects• Heterotropic Allosteric effects• T and R states of cooperative systems• Role of proximal His residue in cooperativity of O2 binding by Hb• Regulation of O2 binding to Hb by bis-phosphoglycerate (BPG)Oxygen Binding to Myoglobin and Hemolobin:Myoglobin binds one O2:Hemoglobin binds four O2:Allosteric Effects and Cooperativity:Allosteric effects occur when the binding properties of a macromolecule change as a consequence of a secondligand binding to the macromolecule and altering its affinity towards the first, or primary, ligand. There need notbe a direct connection between the two ligands (i.e. they may bind to opposite sides of the protein, or even todifferent subunits)• If the two ligands are the same (e.g. oxygen) then this is called a homo-tropic allosteric effect.• If the two ligands are different (e.g. oxygen and BPG), then this is called a hetero-tropic allostericeffect.In the case of macromolecules that have multiple ligand binding sites (e.g. Hb), allosteric effects can generatecooperative behavior. Allosteric effects are important in the regulation of enzymatic reactions. Both allostericactivators (which enhance activity) and allosteric inhibitors (which reduce activity) are utilized to control enzymereactions.Allosteric effects require the presence of two forms of the macromolecule. One form, usually called theT or tense state, binds the primary ligand (e.g. oxygen) with low affinity. The other form, usually calledthe 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 ofligand increases the amount of R state, thus increases the ease of ligand binding.• 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 T state, thusreducing the binding affinity.Biochemistry I, Fall Term Lecture 13 Sept 28, 20052Mechanism of Positive Cooperativityin Hemoglobin:1. Binding of O2 to Fe+2 movesFe+2 atom into the plane ofthe porphyrin ring.2. Movement of Fe+2 pulls theproximal His residue and itsattached helix (F) towardsthe ring.3. Helix F adjusts conformationby rotation of a1b1 dimerrelative to a2b2 dimer.4. Movement of ab dimersalters conformation of Fe+2at unliganded sites throughthe breaking of an extensivenetwork of salt bridges at subunit interfaces.Biochemistry I, Fall Term Lecture 13 Sept 28, 20053Models of Cooperativity in Hemoglobin:Change from T to R states may occur:• In unison , or in a concerted manner(MWC model)• Sequentially (Koshland model)Either of these models fit the experimentaldata well but neither of these models is likelyto be completely accurate in describing the Tto R transition of subunits in hemoglobin.Recent results suggest that the first part ofthe Hb oxygenation reaction occurs inunison, and the remainder occurssequentially.Hetero-tropic Allosteric Effectors in Hemoglobin.There are many hetero-tropic allosteric effectors in Hemoglobin; two examples are:1. Protons: oxygen affinity is decreased at low pH, such as in active muscle that is producing lactic acid. Thisprovides an immediate response to the metabolic state of the tissue.2. BPG: bis-phosphoglycerate binds to the deoxy form of hemoglobin. Therefore it reduces oxygen affinity.This is an adaptive response, requiring several days at high altitude. The production of excess BPG,although it reduces the oxygen affinity, it makes the protein more efficient at delivering oxygen to thetissues.In the above examples, the tense state of hemoglobinbecomes more prevalent than the relaxed state. The allostericeffector stabilizes the tense state, or lowers its energy relativeto that of the relaxed state. Consequently, the oxygen affinityis reduced and the binding curve is shifted to the right, whichenhances oxygen release.The molecular nature of the action of BPG is quite clear:• In deoxy hemoglobin, a positively charged binding pocketexists between two of the four subunits (see Figure 4.26 inCampbell or the Chime page). Thus BPG can easily bind,and when it does so, it stabilizes the deoxy, or tense, formof the protein.• In oxy-hemoglobin, the relative movement of the chains that occurs during the allosteric transition to the Rstate closes this pocket, so BPG can no longer fit.Biochemistry I, Fall Term Lecture 13 Sept 28, 20054Effect of BPG on O2 DeliveryThis graph shows the effect of BPG(bisphosphoglycerate) on the oxygen affinity ofnormal hemoglobin. The level of BPG in theblood at sea level is 2mM. After adaptation tohigh altitudes in 2-4 days the BPG level rises toabout 5 mM.Since BPG binds to the T-state, the bindingaffinity of O2 to hemoglobin is reduced as theBPG concentration increases. (Note: this curve issomewhat different than previous lecture notesbecause it includes the effect of blood pH andsalt on the oxygen affinity).Fractional Saturation pO2(torr)2mM BPG5 mM BPGSea level1000.990.98Rockies 500.930.86Muscle 200.510.32What fraction of O2 is delivered to the tissues at sea level? ([BPG]=2 mM)YpO2=100 = YpO2= 20 = Diff =What fraction of O2 is delivered to the tissues on arrival in Banff? ([BPG]=2 mM).YpO2=50 = YpO2= 20 = Diff =What fraction of O2 is delivered to the tissues after 5 days on the trails? ([BPG]=5 mM)YpO2=50 = YpO2= 20 = Diff =Effect of BPG on O2 Delivery00.10.20.30.40.50.60.70.80.911.10 20 40 60 80 100pO2YHb - 2 mM BPG Hb - 5 mM