Case 3: Carbonic Anhydrase
What types of secondary structure are found in CA II?
Alpha helices, Beta sheets, turns, and loops
Thermodynamics gives us an expression to relate the standard free energy change at pH=7.0 (ΔG°') and Keq. It is: ΔG°' = -RT ln Keq. Characterize ΔG°' under the following conditions (see Table 6.3): Keq = 1 Keq > 1 Keq < 1 (e.g., 0.1)
At Keq = 1 the standard free energy change at pH = 7.0 is equal to 0, the reaction is in a state of equilibrium whereas both the directions of the products and the direction of the reactants are favored. At Keq > 1, ΔG°' is negative, this means that the products are favored at standard conditions. At Keq < 1, ΔG°' is positive, this means that the reactants are favored at standard conditions.
. As shown in Question 7, the Keq for carbonic anhydrase is 1 × 10-3. Explain why H2CO3 is formed in red blood cells near tissues and CO2 is formed near lung using the Le Chatlier's principle and the equation below.
Bicarbonate is formed in the red blood cells near tissues because there is a lesser concentration of bicarbonate than there is carbon dioxide making ΔG is negative, favoring the formation of the products (bicarbonate) following LeChatiler's principle in which the molecule present in lesser concentration will be favored to form. After the red blood cells contain bicarbonate, and they circulate to the lungs, the bicarbonate is now present in a higher concentration than carbon dioxide. Because of the higher concentration of bicarbonate (product) than carbon dioxide (reactant), ΔG is positive, favoring the formation of the reactants following LeChatiler's principle in which the molecule present in lesser concentration will be favored to form.
Blood is buffered with the carbonic acid - bicarbonate buffering system. Name the enzyme that catalyzes the interconversion of CO2 + H2O and H2CO3.
Carbonic Anhydrase
Explain how carbonic anhydrase is affecting the free energy of the reaction in Figure 6.3 in your text book. Be specific and include a discussion of the free energy of reactants, products, and X‡ (the transition state).
Carbonic anhydrase (an enzyme) is affecting the free energy of the reaction by increasing the rate of the reaction, lowering the activation energy. In the presence of carbonic anhydrase, the amount of energy that is needed to reach the transition state is decreased. The enzyme lowers the activation energy, lowering the transition state allowing more substrates reach the transition state and more product to be formed faster.
Using LeChatelier's principle (LCP), explain or illustrate why salicylate-induced hyperventilation causes the pH of the patient's blood to increase.
Hydrogen ion levels increases because carbon dioxide decreased and is no longer an abundant way to make carbonic acid. Because of this, another means to form carbonic acid must be used. Thus, bicarbonate ion and hydrogen ions are used to make bicarbonate, decreasing the hydrogen ion concentration, and increasing the pH. According to LeChatelier's principle, this new way of forming carbonic acid has changed the direction of the reaction because of the decrease in carbon dioxide shifting to form carbon dioxide from bicarbonate ion, which is now being formed from hydrogen ions and the bicarbonate ion.
Blood pH is normally regulated by the respiratory system to control the level of CO2 and by the urinary system to control the level of HCO3- and non-volatile acids in the blood. What effect would Excessive ingestion of baking soda, HCO3- have on blood pH? Explain with LeChatelier's principle.
Increase in bicarbonate ion would increase the blood pH because according to LeChatelier's principle, more carbonic acid will be formed via bicarbonate ion and hydrogen ions. The hydrogen ions are being used up to form carbonic acid, decreasing the concentration of hydrogen ion, ultimately raising the pH in the blood.
How would a deficiency in carbonic anhydrase affect the buffering of blood?
It would lower the concentration of bicarbonate and bicarbonate ion formed in the blood which is used as a buffer to regulate pH. With this deficiency, the pH of the blood would change which can be lethal.
Draw free energy diagrams for the following reactions. Plot reaction progress on the X-axis and free energy on the Y-axis. Label the activation energy. Use a solid line to show the uncatalyzed reaction, and a dashed line to show the catalyzed reaction. Include a key to show what your lines mean. CO2 + H2O --> H2CO3
Non-spontaneous/endothermic
Focus on the following components of the figure: osteoclast, bone, resorptive pit, anion exchanger, H2O+CO2, CA, H+, ATPase. Write a short description explaining how these components fit together to facilitate osteoclast function.
Osteoclast is a site for bone development and restoration. These other components tie into this process by starting with the bone. In order for, bone development to successfully happen some bone must be destroyed first, this happens in the resorptive pit. The acidity of the pit is then achieved through the reaction of water and carbon dioxide with carbon anhydrase as a catalyst. This reaction forms hydrogen ions and carbonate ions to be produced by the dissociation of carbonic acid. The hydrogen ions are pumped out of the cell by the ATPase into the resorptive pit keeping the hydrogen ion concentration high and the pH low. The bicarbonate ion is transferred out of the cell by the anion exchanger.
Bone tissue is rich in cells called osteoclasts. Osteoclasts are rich in CA. In one sentence, summarize the function of osteoclasts.
Osteoclast is a type of bone cell that breaks down bone tissue. It disassembles and digests the composite of hydrated proteins and minerals at a molecular level undergoing bone resorption
Free energy and reaction equilibria are linked. Write the equation that describes the equilibrium constant (Keq) for the chemical reaction catalyzed by carbonic anhydrase: CO2 + H2O ⇌ H2CO3
Products over reactants;
Draw free energy diagrams for the following reactions. Plot reaction progress on the X-axis and free energy on the Y-axis. Label the activation energy. Use a solid line to show the uncatalyzed reaction, and a dashed line to show the catalyzed reaction. Include a key to show what your lines mean. H2CO3 --> CO2 + H2O
Spontaneous
Blood pH is normally regulated by the respiratory system to control the level of CO2 and by the urinary system to control the level of HCO3- and non-volatile acids in the blood. What effect would Holding your breath for an extended period of time have on blood pH? Explain with LeChatelier's principle.
The concentration of carbon dioxide is increasing because it is no longer being expelled from the body. According to LeChatelier's principle, the equilibrium will shift to the right, ultimately increasing the concentration of hydrogen ions and decreases the pH in blood.
Explain why salicylate-induced hyperventilation leads to the values of pO2 and pCO2 symptoms seen in the patient.
The induced hyperventilation leads to the changes in pO2 and pCO2. The body is excessively taking in pO2 and excessively releasing pCO2.
Explain how CA deficiency (e.g., the His 107 --> Tyr mutation) would impact osteoclast function.
The mutation of CA would cause an increase in carbon dioxide and water because it cannot form enough carbonic acid efficiently. Now that there is less carbonic acid accessible, there are no hydrogen ions being pumped into the resorptive pit to keep the pH low, losing the ability to denature proteins. This ultimately osteoclast' function such as bone restoration and bone breakdown cannot function properly.
A genetic analysis of one of the sister's CA II genes indicates that a His to Tyr mutation at amino acid 107 is responsible for the CA deficiency. The His 107 residue is located away from the active site. But its side chain participates in hydrogen bonds, each with a different nearby residue. Explain possible reasons that a mutation to Tyr would lead to problems with CA II structure.
The mutation to Tyr looks like it would not cause any changes because both His and Tyr in this structure can form 2 hydrogen bonding sites, but upon further inspection, this mutation causes major problems. The mutation of His to Tyr at amino acid 107 causes changes in the secondary structure of the loops, ultimately changing the binding site due to Tyrsine's structure.
What is the pH of the resorptive pit? Referencing your answer to 7a, explain why the resorptive pit is acidic, including the role of CA and ATPase.
The pH of the resorptive pit is ~4. The resorptive pit is acidic to denature proteins. For the resorptive pit to stay acidic CA forms bicarbonate ion and hydrogen ions. The hydrogen ions are then pumped into the resorptive pit via the ATPase to keep the pit acidic.
The Keq for carbonic anhydrase is 1 × 10-3. Is the ΔG°' for this reaction zero, negative, or positive? Explain your reasoning. What does that mean about the direction of the reaction under standard conditions?
ΔG°' is positive for this reaction. This happens because the natural log of a number less than 1 is negative which is then multiplied by negative R making ΔG°' positive. This means that the formation of the reactants is favored in this reaction.
Biochemists work with ΔG°'. Define this term and state its units
ΔG°' is the standard free-energy change at pH 7 when the concentration of products and reactants being 1 molar. It is measure in kilojoules or kilocalories.