Problem Set: Fat Metabolism
1. a. How many
14C atoms are incorporated into palmitate when fatty acid synthesis is carried out with the following labelled substrate? Why?
b. What would be the effect on fatty acid synthesis of an increase in intramito- chondrial oxaloacetate levels? How about cholesterol biosynthesis? Briefly explain your answer.
2. a. For the following four reasons, fatty acid biosynthesis is not a simple reversal of fatty acid oxidation:
b. Why is malonyl CoA utilized as the donor of 2-carbon units in fatty acid biosynthesis?
c. Why is it advantageous for the cell to synthesize fatty acids attached to the surface of a single, multifunctional enzyme?
3. Scientists have recently discovered that key eukaryotic proteins, including the oncogene product, ras, undergo a set of post-translational modifications by which mevalonate derivatives are covalently attached to the polypeptide chain. The attachment involves a thioether linkage of a mevalonate derivative to a cysteine residue side chain. Mass spectroscopy has revealed that the mevalonate-derived attachments are either 15 or 20 carbons in length. Given what you know about cholesterol biosynthesis, propose a pathway for the synthesis of these mevalonate-based attachments. (It is not necessary to draw structures--please provide an outline of the biosynthetic strategy).
4. (This is a true story) After sustaining repeated episodes of myocardial infarction, a 6-year old girl homozygous for familial hypercholesterolemia (FH) underwent a combined heart-liver transplant. After surgery, her total plasma cholesterol fell from 1100 mg/dl to the range of 200 to 300 mg/dl, and it remained in that range for the next 13 months. The patient was then treated with low doses of mevinolin, an inhibitor of HMG-CoA reductase, and her cholesterol level fell further to the more normal range of 150-200 mg/dl.
a. What did the transplanted liver furnish to lower her plasma cholesterol levels?
b. Explain how mevinolin contributed to a decrease in her plasma cholesterol.
5. A. Why are phosphoglycerides considered amphipathic molecules?
B. Why do phospholipids form bilayers instead of micelles?
C. What forces drive bilayer formation?
D. Why do phospholipids rarely "flip-flop" from one bilayer leaflet to the other?
6. A. What two properties make triacylglycerols more efficient then glycogen for storage of metabolic energy?
B. How is pyrophosphatase involved in the activation of fatty acids for
b- oxidation?
7. A. Calculate the metabolic energy yield from oxidation of palmitic acid, taking into account the energy needed to activate the fatty acid and transport it into mitochondria.
B. Why are both FAD and NAD
+ used in b-oxidation, rather than just NAD+?
8. What is the physiological role of ketone bodies? Why are they exported from the liver rather than being converted into acetoacetyl-CoA?
9. Explain how nitrogen metabolism influences the risk of diabetic ketoacidosis for an individual with juvenile diabetes.
10. The precise mechanism of ammonia toxicity to the brain is not known. Speculate on a possible mechanism, based on possible effects of ammonia on levels of key intermediates in energy generation.
11. Amino acids are classified as glucogenic, ketogenic, or mixed. Define these classifications and name an amino acid in each group.
ANSWERS TO PROBLEM SET #5:
(1a) Seven; one from each molecule of malonyl CoA which is the activated donor of two-carbon units for fatty acid biosynthesis.
(1b) Increase in citrate levels would increase levels of acetyl-CoA in cytosol because citrate is a carrier of acetyl-CoA from the mitochondrion into the cytoplasm thus stimulating fatty acid synthesis and cholesterol synthesis (since all carbons in fatty acids and cholesterol are derived from cytosolic acetyl CoA)
(2a)
1. Fatty acid biosynthesis requires intermediates that are activated by attachment to acyl carrier protein, not CoA
2. Biosynthesis requires oxidation of NADPH to NADP
3. Fatty acid oxidation occurs in the mitochondrion; synthesis occurs in the cytosol.
4. Fatty acid synthase catalyzes all of the reactions of fatty acid synthesis;
fatty acid degradation is carried out by at least seven separate enzymes.
(b) Decarboxylation of the malonyl CoA makes the condensation of 2, 2-carbon units of acetyl CoA more energetically favorable. Thus, the ATP input required for carboxylation of acetyl CoA to form malonyl CoA actually provides the energy to drive two-carbon unit condensation.
(c) The substrates generated at each round don't diffuse away from the enzyme, and thus, can be generated at a more rapid rate.
3: Mevalonate is the committed, 6 carbon precursor of isopentenyl pyrophosphate, a five carbon precursor of all polyisoprenoid compounds. Cholesterol biosynthesis involves condensation of three isopentenyl pyrophosphate units, which is driven by release and hydrolysis of the pyrophosphate moiety. To attach a farnesyl (or "C-15") group to a cysteine, farnesyl pyrophosphate (directly from cholesterol biosynthesis) may be utilized as the activated donor by a farnesyl transferase enzyme to accomplish the novel modification (no compound names necessary in answer if numbers of carbons are correct). To build a 20 carbon-long unit, one would need a new enzymatic step since cholesterol biosynthesis involves condensation of two 15-carbon precursors, with no 20-carbon-long intermediates. In this case, an enzyme could add 5 carbons to a farnesyl pyrophosphate (using isopentenyl pyrophosphate); this 20-carbon activated "geranyl-geranyl pyrophosphate" compound (it would contain a pyrophosphate group after a "head-to-tail" condensation) could be added by a C-20-specific polyisoprenoid transferase enzyme. Alternatively, the compound could be generated by head-to-head condensation of two gernayl-PPi molecules to yield a similar length (but different structure) compound.
4: a. The transplant provided functional LDL receptors that could clear circulating LDLs. (FH homozygotes lack functional LDL receptors)
b. HMG-CoA reductase is the rate-limiting enzyme of cholesterol biosynthesis. When this enzyme is inhibited, her liver will synthesize less cholesterol, (and the transplanted liver cells will also synthesize more functional LDL receptors).
5: a. the head groups of phosphoglycerides are hydrophilic; their tails are hydrophobic. Thus, the two ends of these molecules have entirely different properties.
b. the two fatty acyl tails of phosphoglycerides are too bulky to form stable micelles; in a micelle, the hydrophobic tails would not be adequately shielded from water molecules.
c. Bilayers are formed by hydrophobic and van der Waals interactions between the fatty acid tails, and electrostatic and hydrogen bonding interactions between the phosphoglyceride head groups and water molecules.
d. The large amount of energy needed to transfer a phosphoglyceride head group across the hydrophobic bilayer core makes this an extremely rare event. All of the types of interactions in answer to C. that keep lipids in a bilayer would need to be disrupted during this transfer.
6. (a) More reduced so the energy yield is greater; they are less hydrated, so that less H
2O needs to be stored along with the energy storage molecule (the organism can weigh less).(b) Pyrophosphatase drives the equilibrium of fatty acyl CoA ligase in favor of the formation of acyl-adenylate, which is then attacked by the re-active sulfhydryl of CoA. The hydrolysis of ATP makes this reaction essentially irreversible, even though the reactant and product are similar in their free energies.
7. Activation of palmitoyl CoA -1 ATP
(or -2 if you include PPi--> 2Pi
oxidation of 8 AcCoA 8 X 10 = 80
oxidation of 7 FAD 7 X 1.5 = 10.5
oxidation of 7 NADH 7 X 2.5 = 17.5
total 106 or 107 ATP
8. Ketone bodies are the preferred energy source of heart muscle, and renal tissue--they are a water soluble source of acetyl units. Under conditions of starvation, fatty acids are broken down to yield AcCoA. However, acetyl CoA cannot enter the citric acid cycle because oxaloacetate is limiting. Under these conditions, ketone bodies are formed in the liver, and exit to peripheral tissues. There, they are converted back into AcCoA. They are exported from the liver because liver lacks succinyl CoA transferase, which is required for acetoacetate generation.
9. Amino acid degradation also generates ketone bodies, in addition to fatty acid oxidation which will lead to ketone body production due to inefficient carbohydrate utilization.
10. One possible theory proposes that ammonia depletes pools of alpha ketoglutarate through the glutamate dehydrogenase and Gln synthetase reactions, which convert alpha KG to Glu and Gln and this diminishes ATP production by reducing flux through the citric acid cycle.
11. Glucogenic: carbons can appear in glucose via gluconeogenesis; ketogenic, generate ketone bodies. Those shaded are both: Glucogenic: Ala, Cys, Gly, Ser, Thr, Trp, Asp, Asn, Phe, Tyr, Ile, Met, Val, Arg, Glu, Gln, His, Pro; Ketogenic: Ile, Leu, Lys, Phe, Trp, Tyr