Question

CHEM 4722/5722 Homework Set #3 (due beginning of class Thu Apr 2)

Homework is to be turned in prior to class and should be done on plain white printer paper. If not, it will be returned for resubmission with a penalty assessed. Word processing should be used for any narrative answers. Calculations and mechanisms can be done freehand but must be very neat, organized, legible and on plain white paper. No lined paper will be accepted.

1. An example of a unsaturated fatty acid is cis-11-heptadecenoic acid. If this compound is oxidized down to CO2 and water via β-oxidation and subsequent pathways, how many ATP equivalents can be obtained? Show all work and list where all of the ATPs come from.

2. Draw out both steps of the malic enzyme mechanism for the conversion of malate to pyruvate. Include nicotinamide moieties, acid/base catalysis, and electron movement.

3. Write a balanced stoichiometric equation for the synthesis of squalene from acetyl-CoA. Indicate the reactions you used to determine your answer and show all work.

4. Refer to your lecture notes or your book for the synthesis of sphingomyelin. Write out the mechanisms (reactant, product structures and electron movement) for each synthetic step to the final product.

5. Mice were divided into four groups, two of which were fed a normal diet and two of which were fed a cholesterol-rich diet. HMG-CoA reductase mRNA and protein levels from liver were measured.

   1. a) What is the effect of cholesterol feeding on HMG-CoA reductase mRNA?

   2. b) What is the effect of cholesterol feeding on HMG-CoA reductase protein?

   3. c) Why is the result in panel B surprising in light of the result in panel A?

   4. d) Suggest possible explanations for the result in panel B.

Answer 1

1. An example of an unsaturated fatty acid is cis-11-heptadecenoic acid. If this compound is oxidized down to CO2 and water via β-oxidation and subsequent pathways, how many ATP equivalents can be obtained? Show all work and list where all of the ATPs come from.

Oxidation of fatty acids into CO2 and water via beta-oxidation and release ATP for cellular functions. Fatty acids with 14 or more carbons (e.g. 17 C as given in question) move into mitochondria through three enzymatic reactions of the carnitine shuttle.

The first reaction is catalyzed by a family of isozymes (acyl-CoA synthetases) present in the outer mitochondrial membrane, which promote the formation of a thioester linkage between the fatty acid carboxyl group and the thiol group of coenzyme A to yield a fatty acyl–CoA, coupled to the cleavage of ATP to AMP and PPi. General reaction is as follows:

Fatty acid + CoA + ATP           Fatty acyl–CoA + AMP + 2Pi

In the second reaction, fatty acids destined for mitochondrial oxidation are transiently attached to the hydroxyl group of carnitine to form fatty acyl–carnitine, catalyzed by carnitine acyltransferase I in the outer membrane. Where the acyl-CoA passes through the outer membrane and is converted to the carnitine ester in the intermembrane space.

In the third and final step of the carnitine shuttle, the fatty acyl group is enzymatically transferred from carnitine to intramitochondrial coenzyme A by carnitine acyltransferase II, which regenerates fatty acyl–CoA and releases it, along with free carnitine, into the matrix. Carnitine reenters the intermembrane space via the acyl-carnitine/carnitine transporter.

Cis-11-hetadecenoic acids (common in the lipids of many plants, some marine organisms) after the transportation into matrix of mitochondria, mitochondrial oxidation takes place into three steps:

Step-1: Fatty acids undergo oxidative removal of successive two-carbon units in the form of acetyl-CoA (further oxidized through citric acid cycle), starting from the carboxyl end of the fatty acyl chain, however, the substrate for the last pass through the beta-oxidation sequence is a fatty acyl–CoA with a five-carbon fatty acid.

Step-2: Five-carbon fatty acids oxidized and cleaved into acetyl-CoA and propionyl-CoA. Again, the acetyl-CoA can be oxidized in the citric acid cycle, but propionyl-CoA enters a different pathway involving three enzymes.

Step-3: Propionyl-CoA is first carboxylated to form the D stereoisomer of methylmalonyl-CoA by propionyl-CoA carboxylase, which contains the cofactor biotin. In this enzymatic reaction, CO2 (or its hydrated ion, HCO3-) is activated by attachment to biotin before its transfer to the propionate moiety, resultant in the formation of D-methylmalonyl- CoA. Formed D-methylmalonyl- CoA epimerized to its L stereoisomer by methylmalonyl-CoA epimerase. The L-methylmalonyl-CoA then undergoes an intramolecular rearrangement to form succinyl-CoA, which can enter the citric acid cycle. This rearrangement is catalyzed by methylmalonyl-CoA mutase, which requires as its coenzyme 5`-deoxyadenosylcobalamin, or coenzyme B12, which is derived from vitamin B12 (cobalamin).

A total of ATP:

C17 – C15 - C13 - C11 – C9 – C7 – C5 = 7 acetyl–CoA + acetyl-CoA and propionyl-CoA

In total: 8 acetyl–CoA + propionyl-CoA

8 Acetyl-CoA + 16O2 + 80Pi + 80ADP         8CoA + 80ATP + 16CO2 + 16H2O

Propionyl-CoA + CO2         succinyl-CoA    FADH2 + NADH

Answer 2. Draw out both steps of the malic enzyme mechanism for the conversion of malate to pyruvate. Include nicotinamide moieties, acid/base catalysis, and electron movement.

Malic enzyme can give rise to pyruvate in the cytoplasm. This enzyme is a decarboxylating enzyme that serves as an additional source of NADPH for lipogenesis. The pyruvate that results from this reaction sequence is then available to reenter mitochondria for conversion to oxaloacetate or acetyl-CoA, or be converted to alanine via ALT. In addition to cytoplasmic conversion of malate to pyruvate, it should be noted that malate can also be shuttled back into mitochondria.

(S)-malate + NADP+    Pyruvate + CO2 + NADPH

Answer 3. Write a balanced stoichiometric equation for the synthesis of squalene from acetyl-CoA. Indicate the reactions you used to determine your answer and show all work.

CH3-COOH CH3-COA  Mevalonate + Isoprene   Squalene

Conclusion

This squalene synthesized from acetyl-CoA further used in the Cholesterol synthesis by addition of isoprene.

Answer-4:

Refer to your lecture notes or your book for the synthesis of sphingomyelin. Write out the mechanisms (reactant, product structures and electron movement) for each synthetic step to the final product

Biosynthesis of sphingolipids: Condensation of palmitoyl-CoA and serine followed by reduction with NADPH
yields sphinganine, which is then acylated to N-acylsphinganine (a ceramide). In animals, a double bond is created by a mixed-function oxidase, before the final addition of a head group: phosphatidylcholine, to form sphingomyelin; glucose, to form a cerebroside.

Palmitoyl-CoA   beta-Ketosphinganine Sphinganine  N-acylsphinganine Ceramide Cerebroside Sphingomyelin

Answer-5:

5. Mice were divided into four groups, two of which were fed a normal diet and two of which were fed a cholesterol-rich diet. HMG-CoA reductase mRNA and protein levels from liver were measured.

a) What is the effect of cholesterol feeding on HMG-CoA reductase mRNA?

b) What is the effect of cholesterol feeding on HMG-CoA reductase protein?

c) Why is the result in panel B surprising in light of the result in panel A?

d) Suggest possible explanations for the result in panel B.

Cholesterol-rich diet feeding affects following factors:

a) From the report, cholesterol-rich diet feeding decrease the synthesis of HMG-CoA reductase mRNA.

b) As per answer of a), it also reduced the expression of HMG-CoA reductase protein.

Conclusion:

Ultimetely, reduced the expression of HMG-CoA reductase following feeding of cholesterol-rich diet suppress the Mevalonate synthesis and and finallty synthesis of cholesterol.