Biochemistry Questions 1. Measuring the concentrations of metaboli...

Biochemistry Questions
1. Measuring the concentrations of metabolic intermediates in a living cell presents great experimental difficulties—usually a cell must be destroyed before metabolite concentrations can be measured. Yet enzymes catalyze metabolic interconversions very rapidly, so a common problem associated with these types of measurements is that the findings reflect not the physiological concentrations of metabolites but the equilibrium concentrations.
A reliable experimental technique requires all enzyme-catalyzed reactions to be instantaneously stopped in the intact tissue so that the metabolic intermediates do not undergo change. This objective is accomplished by rapidly compressing the tissue between large aluminum plates cooled with liquid nitrogen (-190 °C), a process called freeze-clamping.
After freezing, which stops enzyme action instantly, the tissue is powdered and the enzymes are inactivated by precipitation with perchloric acid. The precipitate is removed by centrifugation, and the clear supernatant extract is analyzed for metabolites. To calculate ntracellular concentrations, the intracellular volume is determined from the total water content of the tissue and a measurement of the extracellular volume.
The intracellular concentrations of the substrates and products of the phosphofructokinase-1 reaction in isolated rat heart tissue are given in the table below.

Metabolite                      Concentration
                                              (µM)
Fructose 6-phosphate          87.0
Fructose 1,6-bisphosphate    22.0
ATP                                       11,400
ADP                                       1,320

a. Calculate Q, [fructose 1,6-bisphosphate][ADP]/[fructose 6-phosphate][ATP], for the PFK1 reaction under physiological conditions.
b. Given a ΔG’° for the PFK-1 reaction of -14.2 kJ/mol, calculate the equilibrium constant for this reaction.
c. Compare the values of Q and K’eq. Is the physiological reaction near or far from equilibrium? Explain. What does this experiment suggest about the role of PFK-1 as a regulatory enzyme?

2. Given the following information for the isocitrate dehydrogenase reaction: [NAD+ ]/[NADH] = 9, [α-ketoglutarate] = 0.1 mM, [isocitrate] = 0.02 mM, assume standard conditions for CO2.
a. Look up the E’° for the appropriate half-reactions in your text and calculate the value of ΔG’° based on those values.
Determination of the reduction potentials for this enzyme is covered in one of the supplemental readings. You should read through the supplemental article on Isocitrate
Dehydrogenase and see the lengths to which one must go in order to determine accurate physical quantities.
b. Is this reaction a likely site for metabolic control? Explain.
c. The ΔG’° is frequently listed as -21 kJ·mol-¹. Comment on the difference from the value that you calculated in part a).

3. For each of the following questions, assume only one cycle of the TCA is completed during the course of the experiment.
a. Oxaloacetate labeled at C4 with the radioactive isotope ¹⁴C is added to a suspension of respiring mitochondria. What is the fate of the labeled carbon?
b. In a separate experiment, Acetyl~CoA labeled at C1 of the acetyl group with ¹⁴C is added to a suspension of respiring mitochondria. What is the fate of the labeled carbon?

4. Reaction 8 (ΔG’° = +29.7 kJ/mol) and Reaction 1 (ΔG’° = -32.2 kJ/mol) of the citric acid cycle are considered to be coupled, because the exergonic cleavage of the thioester bond of acetyl-CoA in Reaction 1 drives regeneration of oxaloacetate in Reaction 8.
a. Write the balanced equation for the overall coupled reaction.
b. Calculate the ΔG’° for the overall coupled reaction.
c. What are the equilibrium constants for the individual reactions? (at 25°C)
d. What is the equilibrium constant for the combined or coupled reaction? (at 25°C)

5. The reduction of pyruvate to lactate is an extremely important reaction that occurs in muscle when they are working anaerobically, such as during brief sprints.
As noted in class, the purpose of this pathway is to replenish NAD+ (i.e. reoxidize NADH to NAD+ ) and allow further glycolysis.
The half-reactions involved in the lactate dehydrogenase (LDH) reaction and their standard reduction potentials are:

Pyruvate      + 2H+ 2e- -----> lactate E’° = -0.185 V
NAD+          + 2H + 2e- ----------> NADH + H+ E’° = -0.320 V

For simplicity, the following calculations are all to be done at 25°C. As we have seen earlier, the adjustment to 37°C is minor.
a. Calculate the ΔG’ for the reaction at 25 °C under each of the following conditions:
i. [lactate]/[pyruvate] = 1; [NAD+]/[NADH] = 1
ii. [lactate]/[pyruvate] = 180; [NAD+]/[NADH] = 180
iii. [lactate]/[pyruvate] = 1000; [NAD+ ]/[NADH] = 1000

Hint: Start by calculating E’ for each half-reaction at the specified reactant and product ratios. Pay attention to what is specified. Pay attention to units and scale factors (e.g. milli, micro, kilo, mega, …)! Use the calculated E’ values to determine ΔE’ and use that to calculate ΔG’.
b. Under what conditions (in terms of [lactate]/[pyruvate] and [NAD+]/[NADH] ratios together, i.e. in terms of Q for the complete reaction) will the reaction favor spontaneous NADH oxidation?
Hint: For this question you will need to consider the complete reaction (combined halfreactions) and how the ratios combine in that setup (i.e. the value of Q).

c. Here, you will tie the previous result into the “big picture” of glycolysis.
In order for the free energy change of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) reaction to favor glycolysis, the [NAD+ ]/[NADH] ratio must be maintained close to 103. As indicated above, under anaerobic conditions in mammalian skeletal muscle, lactate dehydrogenase performs this function. How high can the [lactate]/[pyruvate] ratio become in muscle cells before the LDH-catalyzed reaction ceases to be favorable in the direction of NAD+ production while still maintaining the desired [NAD+]/[NADH] ratio of 1000?