BCH Graduate Survey of Biochemistry

Transcription

1 BCH 5045 Graduate Survey of Biochemistry Instructor: Charles Guy Producer: Ron Thomas Director: Glen Graham Lecture 12 Slide sets available at:

2 David L. Nelson and Michael M. Cox LEHNINGER PRINCIPLES OF BIOCHEMISTRY Fifth Edition CHAPTER 5 Protein Function 2008 W. H. Freeman and Company

3 The binding of oxygen by the iron of the heme group is forced to be at an angle because of steric hindrance, while carbon monoxide is slightly smaller and not sterically hindered and as a result carbon monoxide binds much more tightly to the heme group binding site of myoglobin than does oxygen.

4 Notice the high degree of 3-dimensional structural similarity between myoglobin and hemoglobin. Myoglobin functions as a single independent polypeptide, but hemoglobin has four polypeptide subunits in the holoprotein.

5 Dominant interactions occur between hemoglobin subunits and the strongest subunit interactions occur between alpha one and beta one subunits change only modestly while the alpha one and beta two subunits contact interactions are much larger.

6 Hemoglobin has two affinity states for the ligands it binds. Interaction between His HC3 and Asp FG1 and Lys C5 are important to the T state of deoxyhemoglobin, or Tense binding state. There is also an R state.

7 Several ion pairs are important in helping to stabilize the T state of deoxyhemoglobin which is the low affinity ligand binding state.

8 A shift in the position of the F helix when the heme binds O 2 is considered to be a change that triggers the T R transition.

9 The oxygen binding sites on hemoglobin can shift from low-affinity to a high-affinity state. This occurs in part because of the cooperative binding properties within and between the subunits of the hemoglobin protein of the that is indicated by a sigmoid binding curve. In the high affinity state, hemoglobin is more sensitive to the small differences in oxygen concentration between the tissues and the lungs.

10 This figure illustrates the principle of cooperativity and cooperative ligand binding in the context of protein 3- dimensional structure and the role conformational changes in the 3-dimensional structure play in the changing binding affinity of the protein for the ligand.

11 Reversible binding by more than one ligand binding site can be described by the simple equation: P + nl PL n And this can be characterized by an equilibrium where K a = [PLn]/[P][L] n where K a is the association constant. Θ = [L] n /([L] n + K d ) Take the log of both sides, rearrange and you arrive at the Hill equation: Log(Θ/1- Θ) = n log [L] log K d where K d = [L] n 0.5 and the slope of the Hill plot is n H which is a measure of the degree of cooperativity. The n H value can either positive or negative.

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13 Symmetry and Sequential Models to Explain Binding and Cooperativity

14 This slide is not in the lecture video The Symmetry model or Concerted model or MWC model or two-state model assumes that subunits in a cooperative binding protein are all identical with at least two functional conformations that are in a state of equilibrium with each other. In the case of hemoglobin, the R and T conformations. Transition from one conformation to another is not dependent on ligand binding or dissociation, but instead the K a and K d reflect the affinity of the ligand binding site of each conformation independent of whether the ligand is bound or not. The binding or release of the ligand thus causes a global shift in the equilibrium between the two conformations for all binding sites, but does not cause of the conformational change. In contrast, the Sequential model proposed by Koshland, Nemethy and Filmer is based on the concept of induced fit, where ligand binding actually causes a conformational change in the subunit. Such a conformational change then influences a second subunit to undergo a similar change in conformation that influences its ligand binding site and so on. The MWC model seems to be the most tested and supported by the accumulated body of experimental evidence, although there are circumstances where it fails to predict all experimental outcomes.

15 The ph of the solution can influence the oxygen or ligand binding characteristics of hemoglobin. What part of hemoglobin is being affected by ph?

16 Θ, is the proportion of binding sites with oxygen bound is sensitive to ph. The ph of the blood in the lungs is 7.6 and in the tissues, 7.2. So what is the effect of low ph on the binding of oxygen by hemoglobin? Hemoglobin transports about 40% of H + and about 20% of the CO 2 formed in tissues to the lungs the rest is transported as HCO 3. The effects of CO 2 and ph on the binding of oxygen by hemoglobin is known as the Bohr effect.

17 Another effector of oxygen binding by hemoglobin is the metabolite BPG. It is a byproduct of glycolysis occurring in tissues at increasingly lower oxygen levels. We have about 5 mm concentration of BPG, but people living in Denver have about 8 mm in their blood. So what does this diagram tell you about oxygen binding by hemoglobin? Note the change in structure in panels b and c where the one molecule of BPG will bind in a pocket between the β subunits. Where does BPG bind relative to oxygen and carbon dioxide?

18 Sickle cell anemia would be the first genetic disease to be described at the molecular level. Pauling s group was the first to show that the sickle (S) hemoglobin had a charge difference from that of other hemoglobins in 1949, and because of this he called the anemia a molecular disease. There are many allelic variants of hemoglobin in humans. Most are of little structural or functional significance. V. M. Ingram s group found in Sickle cell anemia there is a single amino acid substitution from a Glu to Val or E6V of the β-chain. This one change when the hemoglobin becomes deoxygenated, causes S hemoglobin to become insoluble and form long rods. The RBC becomes sickle shaped. Why is it unwise for someone with this trait to participate in sports? What is the biochemical explanation why the E6V change causes the hemoglobin to form large insoluble rods?