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1. What is the site to which the allosteric inhibitor binds?
It is a specialized regulatory site; upon binding the inhibitor, the enzyme changes its shape slightly. The shape change includes the remote region encompassing the active site of the enzyme, and is sufficient to prevent the catalysis from taking place at the active site.
2. How does an enzyme facilitate chemical reactions?
An enzyme facilitates chemical reactions by bringing the substrates in close proximity, and lowering the activation energy of the chemical reaction.
3. List four factors that influence the rate of a chemical reaction.
State whether increasing the factor will increase or decrease the rate of reaction.
Chemical reaction rate can be regulated by changing the temperature (increase), the substrate concentration (increase), the enzyme concentration (increase) or the enzyme activity (increase).
4. What is the nature of protein-ligand binding?
What is the nature of modulator-protein binding?
Protein-ligand binding are electrical or Wan- der Waals in nature (non-covalent).
Modulators proteins can bind either non-covalently, or covalently.
1. Hydrolysis of ATP yields the right amount of energy for many cellular processes. List some cellular processes that use ATP as their energy source
- protein synthesis
- Na-K ATPase
- Ca ATPase
- actomyosin ATPase
2. ATP is not abundant in cells. Answer the following questions to estimate how long the ATP stored in your brain would support its activity, assuming ATP is the only energy source.
a. Estimate the brain’s ATP energy store, assuming that [ATP] = 5 mM in brain cells, that the free energy change on hydrolysis of ATP (ΔGo’ ) is -7.3 kcal/mol ≈ -30.5 kJ/mol and that the mass of the brain is 1. kg.
- 5 mM * (-30.5 kJ/mol) * (2/3 L intracell water/L tissue)
- * (1 L tissue/kg tissue) ≈100 Joules/kg tissue.
Brain’s ATP Supply: 100 J/kg * 1.5 kg = 150 J
b. Estimate the rate at which the brain uses energy, assuming that it uses 15% of the energy that your whole body uses, and that the rate of energy use by your body is 100 watts.
15 W = 15 J/s
c. In how long would the brain’s ATP store (from part a) be completely depleted at the rate you calculated in part (b).
Time to use ATP supply (at rest): (150 J)/(15 J/s) = 10 s
3. a. Explain why the steps that are controlled in metabolic pathways are typically those with a large negative ΔG.
A large negative ΔG means that the free energy of reactants is much greater than the free energy of products. The reaction would occur spontaneously if there were no activation barrier, but typical biochemical reactions have activation barriers large enough to keep reactions from occurring at significant rates in the absence of enzymes. Enzymes catalyze biochemical reactions by taking advantage of binding energy between enzyme and its substrate, lowering activation barriers.
- A large drop
- in potential energy gives an enzyme opportunity to control a reaction whose net
- rate can be small or large. Control over a reaction that has only a small
- change in free energy could not appreciably modulate its net rate. Also, a
- large drop in free energy means that the reverse reaction is unlikely to occur,
- so the reaction is a ‘committed step’ – lack of control might waste free
b. ATP is a substrate of the enzyme PFK. Typically, increasing the concentration of the reactants in a chemical reaction increases
the rate of the reaction. On the contrary, increasing [ATP] slows the reaction catalyzed by PFK. Explain.
There are two binding sites for ATP on PFK, one an active site where ATP is hydrolyzed in order to allow phosphorylation of fructose 6 phosphate, and another at which ATP binds as an allosteric modulator. The allosteric modulator changes the PFK to lower the rate of the controlled reaction.
4. The lactate shuttle hypothesis is that when ATP demand is high in a cell, glycolysis proceeds rapidly, producing pyruvate faster
than it can be converted to acetyl CoA. The excess pyruvate is converted to lactate, which is then shuttled to tissues that oxidize it (by first converting it back to pyruvate, which feeds the Krebs cycle and Ox Phos.)
a. In a cell that is performing glycolysis rapidly, the comversion of pyruvate to lactate is catalyzed by the enzyme lactate dehydrogenase
pyruvate + NADH + H+ à lactate + NAD+
Write the reaction that must occur in a cell that hosts oxidation of lactate, assuming that LDH is also the catalyst there. Under what
cellular conditions might the reaction be favorable in either type of cell?
Lactate + NAD+à pyruvate + NADH + H+
In the lactate producing cell, high [NADH] and [H+] would be favorable. In the lactate receiving cell, high [NAD+] would be favorable.
b. Brooks and colleagues found that lactate was produced by fast-twitch skeletal muscle, and consumed (through Krebs cycle and oxidative
phosphorylation) by slow-twitch skeletal muscle and cardiac muscle. How could oxidation of lactate be measured?
They labeled lactate with radioactive carbons, and measured production of radioactive CO2
5. a. Control of PDC
a. A phosphatase and kinase regulate activity of the pyruvate dehydrogenase complex (as shown on slide 37 of today’s lecture). The
kinase inactivates PDC. Which of the modulators of the kinase shown in the figure provide negative feedback?
All of them
b. The phosphatase that activates PDC is itself activated by Ca++. What kind of control does calcium help provide.
Feedforward control – high calcium concentration signals activation of some cells (e.g. muscle cells), and activation will lead to greater need of ATP.
c. The enzymes in PDC are also allosterically regulated. ATP, acetyl CoA and NADH inhibit PDC enzymes, and AMP, CoA and NAD+allosterically activate PDC enzymes. What kind of control is exerted by these allosteric modulators?
Negative Feedback Control
6. NADH inhibits two of the enzymes in the Krebs (TCA) cycle. Explain why this inhibition might be useful.
Viewing NADH as an output of the TCA cycle, negative feedback might regulate the speed of the cycle.
6. The rate of Ox Phos is usually controlled by [ADP]. Explain how this ‘acceptor control’ regulates [ATP].
As ATP is hydrolyzed, ADP is produced. Increased [ADP] causes more Ox Phos, which produces more ATP.
1. Explain what happens to red blood cells in hypotonic, isotonic and in hypertonic solutions.
Isotonic: The solutions being compared have equal concentration of solutes.
Hypertonic: The solution with the higher concentration of solutes.
- Hypotonic: The solution with the lower concentration of solutes.
- When red blood cells are placed in a 0.9% salt solution, they neither gain nor lose water by osmosis. Such a solution is said to be isotonic.
- Blood serum is isotonic with respect to the cytoplasm, and red cells in that solution assume the shape of a biconcave disk.
If the concentration of water in the medium surrounding a cell is greater than that of the cytosol, the medium is said to be hypotonic. Water enters the cell by osmosis. A red blood cell placed in a hypotonic solution (e.g., pure water) bursts immediately ("hemolysis") from the influx of water.
If red cells are placed in hypertonic solution they lose water by osmosis and the cells shrivel up.
2. What is the chemical basis to that phenomenon?
Osmosis is the net movement of water across a selectively permeable membrane driven by a difference in solute concentrations on the two sides of the membrane. A selectively permiable membrane is one that allows unrestricted passage of water, but not solute molecules or ions.
3. What are the different mechanisms that cell use to move molecules across the plasma membrane along the electro-chemical gradient?
The mechanism the cell use to move molecules along the gradient is simple diffusion or facilitated diffusion.
- Facilitated diffusion takes of ions place through proteins, or assemblies of proteins, embedded in the plasma membrane. These transmembrane proteins form a water-filled channel through which the ion can pass down its concentration gradient.The transmembrane channels that permit facilitated diffusion can be opened or closed. They are said to be "gated". Some types of gated ion channels are ligand-gate, mechanically-gated,
- and light-gated.
4. What are the different mechanisms that cell use to move molecules across the plasma membrane against the electro-chemical gradient?
Active transport is the pumping of molecules or ions through a membrane against their concentration gradient. It requires some form of energy. Depending on the source of energy used they will be termed direct or indirect active transport:
Direct Active Transport. Some transporters bind ATP directly and use the energy of its hydrolysis to drive active transport.
Indirect Active Transport. Other transporters use the energy already stored in the gradient of a directly-pumped ion.
Direct active transport of the ion establishes a concentration gradient. When this is relieved by facilitated diffusion, the energy released can be harnessed to the pumping of some other ion or molecule.