1. People oxidize ethanol in their liver. There, ethanol is oxidized to acetaldehyde, with NAD⁺ serving as the electron acceptor. (Subsequently, acetaldehyde is oxidized to acetate, then to acetyl CoA, and finally to carbon dioxide via the citric acid cycle.) Oxidation of ethanol causes the intracellular ratio of NADH to NAD⁺ to rise.

In malnourished people, or healthy people who have missed several meals, drinking even moderate amounts of ethanol significantly lowers blood glucose. The liver synthesizes less glucose than normal. Moreover, the intracellular ratio of lactate to pyruvate increases. By contrast, ethanol taken by a healthy, well-fed person whose liver contains normal amounts of glycogen maintains normal blood glucose, and glucose production by the liver remains close to normal. Nevertheless, the intracellular ratio of lactate to pyruvate increases.

a) How do elevated intracellular ratios of NADH to NAD⁺ from ethanol lead to an increase in the ratio of lactate to pyruvate?

b) Briefly describe how an increase in the ratio of lactate to pyruvate would impair gluconeogenesis in the liver of a malnourished individual.

c) Why is the blood-glucose-depressing effect of ethanol less when the liver has a normal amount of glycogen?

2. Stimulation of Oxygen consumption by Oxaloacetate and Malate. Early investigators observed that the addition of small amounts of oxaloacetate or malate to suspensions of pigeon-breast muscle cells (a very actively respiring tissue) stimulated the oxygen consumption of the tissue. Surprisingly, when the amount of oxygen consumed was measured, it was about seven times more than the amount necessary to oxidize the added oxaloacetate or malate completely to carbon dioxide and water.

(a) Why does the addition of oxaloacetate or malate stimulate oxygen consumption?

(b) Why is the amount of oxygen consumed so much greater than the amount necessary to oxidize the added oxaloacetate or malate completely?

3. Phosphofructokinase (PFK) catalyzes the formation of fructose 1,6-bisphosphate from fructose 6-phosphate, using ATP as a phosphoryl donor. As an important regulatory enzyme in the glycolytic pathway, PFK is influenced by a number of small molecule metabolites. Describe how the specified levels of the two metabolites listed below would affect the activity of liver PFK, and explain how the effect is consistent with the normal function of the liver.

a) High levels of ADP.

b) Low levels of fructose 2,6-bisphosphate (F-2,6-P2). Please include in your explanation a description of the way changes in blood glucose concentration lead to a decrease in levels of F-2,6-P2.

4. Sublethal cyanide poisoning may be reversed by the administration of nitrites. These substances oxidize hemoglobin, which has a relatively low affinity for CN-, to methemoglobin, which has a relatively high affinity for CN-. Why is this treatment effective?

5. The number of molecules of oxaloacetate in a mitochondrion. In the last reaction of the tricarboxylic acid cycle, malate is dehydrogenated to regenerate the oxaloacetate necessary for the entry of acetyl-CoA via the citrate synthase reaction:

[oxaloacetate] [NADH]

K'eq = ---------------------------

[malate] [NAD⁺]

The measured concentration of malate in rat liver mitochondria is about 0.2 mM when the NAD⁺/[NADH] ratio is 10. Calculate the concentration of oxaloacetate in these mitochondria.

(c) Rat liver mitochondria are roughly spherical, with a diameter of about 2 µm. To appreciate the magnitude of the oxaloacetate concentration in mitochondria, calculate the number of oxaloacetate molecules in a single rat liver mitochondrion.

6. Submitochondrial particles are large pieces of the inner mitochondrial membrane that close inside out to form spherical membrane vesicles. These particles can synthesize ATP, if NADH is added. Draw diagrams that illustrate:

a) how and where electron transport from NADH and subsequent proton transport occur in these particles. Where will the ATP be found - inside or outside the vesicles?

b) whether ADP, or whether O₂ will be required for ATP synthesis.

c) how dinitrophenol, a weak organic acid, allows electron transport without ATP synthesis.

7. Degree of reduction of electron carriers in the respiratory chain. The degree of reduction of each electron carrier in the respiratory chain is determined by the conditions existing in the mitochondrion. For example, when the supply of NADH and O₂ is abundant, the steady-state degree of reduction of the carriers decreases as electrons pass from the substrate to O₂. When electron transfer is blocked, the carriers before the block become more reduced while those beyond the block become more oxidized (Syllabus, XV A.4.). Predict the state of oxidation of each of the following carriers in the respiratory chain (ubiquinone and cytochromes b, c1, c, and a), for each of the conditions below.

(a) Abundant supply of NADH and O₂ but cyanide added

(b) Abundant supply of NADH but O₂ exhausted

(c) Abundant supply of O₂ but NADH exhausted

(d) Abundant supply of NADH and O₂

8. How many protons in a mitochondrion? Electron transfer functions to translocate protons from the mitochondrial matrix to the external medium establishing a pH gradient across the inner membrane, the outside more acidic than the inside. The tendency of protons to diffuse from the outside into the matrix, where the [H⁺] is lower, is the driving force for ATP synthesis via the ATP synthase. During oxidative phosphorylation by a suspension of mitochondria in a medium of pH 7.4, the internal pH of the matrix has been measured as 7.7.

(a) Calculate [H⁺] in the external medium and in the matrix under these conditions.

(b) What is the outside:inside ratio of [H⁺]? Comment on the energy inherent in this concentration ratio.

(c) Calculate the number of protons in a respiring liver mitochondrion, assuming its inner matrix compartment is a sphere of diameter 1.5 µm.

(d) From these data would you think the pH gradient alone is sufficiently great to generate ATP?

(e) If not, can you suggest how the necessary energy for synthesis of ATP arises?

9. Stereochemistry.

(a) Draw glucose in its extended (open) form. Indicate which stereocenters are in the R configuration, and which are in the S configuration.

(b) Draw the nicotinamide ring of NADH, indicating which proton is above the plane of the ring and which proton is below the plane. Identify the ProR proton.

10. Pentose Phosphate Pathway. See Lecture Notes pg. 38.

(a) Explain how the following transformation:

5 Glucose 6-phosphate + ATP ---> 6 Ribose 5-phosphate + ADP + H⁺

can be accomplished using the reactions of glycolysis and the nonoxidative branch of the pentose phosphate pathway.

(b) Explain how the transformation:

Glucose 6-phosphate + 12 NADP⁺ + 7 H₂O --->

6 CO₂ + 12 NADPH + 12 H⁺ + P_i

can be accomplished using the reactions of glycolysis and and both branches of the pentose phosphate pathway.