MT1 Material Flashcards
C18:1 (Δ9) cis-9-Octadecenoic acid
Also an omega-9 fatty acid
C20:5 (Δ5,8,11,14,17) Eicosapentaenoic acid (EPA)
Also an omega-3 fatty acid
Palmitic acid
C16:0
Oleic acid
C18:1 (Δ9)
Stearic acid
C18:0
Linoleic acid
C18:2(Δ9,12)
Linolenic acid
C18:3(Δ9,12,15)
Arachidonic acid
C20:4 (Δ5,8,11,14)
Triacylglycerol
Glycerol
What is the general structure of a glycerophospholipid?
Serine
Ethanolamine
Choline
Glycerol
Inositol
If X = H, name the following:
1-palmitate-2-linoleate-phosphatidic acid
What is the name of the head-group substituent for phosphatidic acid?
H
What is the name of the head-group substituent for phosphatidylethanolamine?
Ethanolamine
What is the name of the head-group substituent for phosphatidylcholine?
Choline
What is the name of the head group substituent for phosphatidylserine?
Serine
What is the name of the head-group substituent for phosphatidylglycerol?
Glycerol
Phosphatidylinositol
Cardiolipin
Phosphatidylglycerol
(head-group substituent of cardiolipins)
If X = H, name the following:
Oleic acid ceramide
and
Oleate ceramide
(both accepted as correct answers)
This molecule is a…. sphingolipid!
If the head group substituent is -H, what is the name of the sphingolipid?
Ceramide
What is the name of the head-group substituent?
What is the name of the resulting sphingolipid?
C26:0 sphingomyelin
What is the name of the head-group substituent?
What is the name of the resulting sphingolipid?
Glucose
Glucosylcerebroside
A cerebroside
What is a ganglioside?
A sphingolipid with a head-group substituent that is a complex oligosaccharide.
What affects melting point of FA?
Chain length: more carbons > higher melting T due to more van der Waals interactions
Number of double bonds: more double bonds > lower melting T (cis double bonds introduce kinks in FA tails disrupting van der Waal’s interactions)
What is the overall pathway for FA degradation? [4]
What is the overall pathway for FA synthesis? [4]
Malonyl-CoA
Tyrosine
Phosphatidyl inositol
Ceramide
Stearate (C18:0) esterified to wax: long chained alcohol (C18:1 Δ9)
Squalene
Choline
Trans oleic acid (C18:1 trans Δ9)
Linoleoyl carnitine
Bile salt: taurocholic acid
Acetone
p-hydroxyphenylpyruvic acid
(phenylalanine would only receive partial marks - see structure of phenylalanine below)
farnesyl pyrophosphate
ceramide esterified with an oleate derived acyl chain
Phosphatidylinositol esterified with stearate and arachidonate dervied acyl chains
Glycerol
Urea
ß-ketostearoyl-CoA
Phosphatidylinositol with palmitate and arachidonate derived acyl chains
alpha-ketoacid of isoleucine
or
2-keto-3-methylpentanoate
or
alpha-keto-ß-methylpentanoate
Carnitine
A ketogenic amino acid is one which is degraded to:
acetyl-CoA or acetoacetyl-CoA
Acetoacetyl-CoA
Acetyl-CoA
Phosphatidylethanolamine esterified with two stearic acids (C18:0).
Sphingomyelin with an amide linkage to palmitic acid (C16:0)
A glucogenic amino acid is one which is degraded to:
pyruvate or TCA cycle intermediates
Oxidative deamination is the conversion of an amino:
a. group from an amino acid to a keto group requiring NAD(P)H
b. acid to a carboxylic acid plus ammonia
c. acid to a keto acid plus ammonia
d. group from an amino acid to a carboxylic acid
b.
Oxidative deamination is the conversion of an amino acid to a keto acid plus ammonia.
During severe starvation, carbon from each of the following two substrates: an odd-numbered saturated acid AND tyrosine, could be converted to:
a. glucose
b. CO2
c. ketone bodies
d. propionyl CoA
e. A and B
f. A, B and C
g. All of the above
f. A, B and C
Where does urea synthesis take place primarily?
In tissues of the liver.
What are the essential amino acids?
Jim has been eating a very monotonous (i.e. unvaried) diet. If Jim’s urine contains unusually high concentrations of urea, which one of the following diets has he probably been eating recently?
a. High carbohydrate, very low protein
b. Very high carbohydrate, no protein, no fat
c. Very very high fat, high carbohydrate, no protein
d. Very high fat, very low protein
e. Very low carbohydrate, very high protein
f. None of the above, Jim suffers from an untreated urea cycle enzyme disorder.
e.Very low carbohydrate,very high protein
Glutamine synthetase converts […] to […] whereas glutamate dehydrogenase converts […] to […].
Glutamine synthetase converts glutamate to glutamine whereas glutamate dehydrogenase converts glutamate to alpha-ketoglutarate.
Nonessential amino acids are amino acids other than those required for protein synthesis.
True or false.
False
Nonessential amino acids are not utilized by mammalian proteins.
True or false.
False
Nonessential amino acids are synthesized by plants and bacteria, but not by humans.
True or false?
False
Nonessential amino acids can be synthesized in humans as well as in bacteria.
True or false?
True
Nonessential amino acids may be substituted with other amino acids in proteins.
True or false?
False
An amino acid that does NOT derive its carbon skeleton, at least in part, from alpha-ketoglutarate is:
a. arginine
b. glutamate
c. glutamine
d. proline
e. lysine
The neurotransmitter dopamine is an intermediate in the conversion of:
a. phenylalanine to homogentisate
b. phenylalanine to tyrosine
c. tyrosine to epinephrine
d. tyrosine to phenylalanine
e. tyrosine to phenylpyruvate
Dopamine is an intermediate in the conversion of tyrosine to epinephrine.
The fatty acid elongation system in mammals involves the same four-step sequence seen in the fatty acid synthase complex.
True or false?
True
One round of reactions catalyzed by elongases (fatty acid elongation) require 2 NADPH to extend the chain by 2 carbons.
True or false?
True.
Regarding elongases, the reactions produce stearoyl-CoA by the extension of palmitoyl-CoA.
True or false?
True.
Malonyl-CoA is used as a substrate in fatty acid elongation by elongases.
True or false?
True.
Regarding elongases, the immediate precursor of the added carbons is acetyl-CoA.
True or false?
False
(True in fatty acid synthesis)
In mammalian cells, what feeds fatty acid synthase its reducing power?
NADPH is produced in the cytosol by malic enzyme.
Note: NADPH is also produced by pentose phosphate pathway.
Acetyl-CoA is transported out of the mitochondrion via the citrate shuttle.
True or false?
False.
Oxaloacetate + acetyl-CoA > citrate
Coenzyme A is not transported across the mitochondrial membrane.
True or false?
True.
There are two separate pools of coenzyme A.
Where is malonyl-CoA formed?
Malonyl-CoA is formed in the cytosol by acetyl-CoA carboxylase (ACC).
Oxaloacetate is transported through OAA transporter from the matrix to the cytosol allowing fatty acid synthesis to proceed.
True or false?
False, carbon returns via malate or pyruvate.
What is cholesterol synthesized from?
acetyl-CoA
Which metabolite regulates the activity of carnitine acyl transferase I (CAT I) ?
malonyl-CoA
Atrophy is defined as a decrease in the size of a tissue due to cellular shrinkage commonly observed during starvation. The decrease in cell size is caused, in part, by the loss of proteins. In skeletal muscle, the ubiquitin-proteasome system acts for example on sarcomeric proteins such as actin, myosin, troponin, and tropomyosin - all necessary for muscle contractions.
What is the overall (big picture) purpose of ubiquitin-proteasome system on sarcomeric proteins under starvation conditions?
During starvation the body lacks fuel sources and needs to break down its non-essential protein reserves into amino acids. Skeletal cells contain the vast majority of the body’s protein reserves. The ubiquitin-proteasome system breaks down the intercellular proteins. The 20 amino acids released from muscle then serve as precursors to make glucose (gluconeogenesis by liver), make ketone bodies (also liver) OR their carbon skeletons are oxidized directly. Oxidation of these fuels produces reducing agents (e.g. NADH) driving the ETC, generating an electrochemical gradient, thus allowing ATP synthase to make ATP.
Atrophy is defined as a decrease in the size of a tissue due to cellular shrinkage commonly observed during starvation. The decrease in cell size is caused, in part, by the loss of proteins. In skeletal muscle, the ubiquitin-proteasome system acts for example on sarcomeric proteins such as actin, myosin, troponin, and tropomyosin - all necessary for muscle contractions.
Under starvation conditions, muscle cells do not completely ‘vanish’ as the majority of other cytoplasmic ‘house keeping’ proteins (e.g. the glycolytic enzyme aldolase) remain at a stable concentration. Explain this apparent contradiction.
The ubiquitin-proteasome system is specific!
Ubiquitination is targeted at a much higher rate at sarcomeric proteins. The cell will contain specific E3s that will preferentially target sarcomeric protein over other house-keeping proteins. Other proteins such as metabolic proteins/enzymes that the cell needs to survive will be maintained (i.e. the rate of degradation will equal rate of synthesis).
Atrophy is defined as a decrease in the size of a tissue due to cellular shrinkage commonly observed during starvation. The decrease in cell size is caused, in part, by the loss of proteins. In skeletal muscle, the ubiquitin-proteasome system acts for example on sarcomeric proteins such as actin, myosin, troponin, and tropomyosin - all necessary for muscle contractions.
What proteins do you expect will increase in concentration under these starvation conditions? Why?
Proteins involved in protein degradation, such as ubiquitin, E1 (Ub activating enzyme), E2 (UB conjugating enzyme) and the specific E3 (Ub ligase) required to target each of the sarcomeric proteins, as well as the subunits (e.g. alpha and ß) required to synthesize the 26S proteasome. In the liver cell the urea cycle enzymes will be upregulated in order to dispose of the nitrogenous waste.
oxaloacetate
Pyruvate
What reaction does pyruvate carboxylase catalyze?
Pyruvate
Tyrosine
Tyrosine
Phenylalanine
Glutamate
Draw glutamate.
Glutamate
Glutamate
Leucine
Leucine
Glutamate
Alpha-ketoglutarate
α-ketoglutarate
α-ketoglutarate
Hydroxyphenylpyruvate
4-Hydroxyphenylpyruvate
These two substrates and alanine aminotransferase yields…
Pyruvate and glutamate
Tyrosine aminotransferase and the following substrates will yield…
Hydroxyphenylpyruvate and glutamate
Branched chain aminotransferase and these two substrates will yield…
Leucine and alpha-ketoglutarate
Aspartate aminotransferase and these two substrates will yield…
Oxaloacetate and glutamate
Tyrosine aminotransferase and these two substrates will yield…
Hydroxyphenylpyruvate and glutamate
Branched chain aminotransferase can use alpha-ketoisocaproate as shown. What is the metabolic fate of alpha-ketoisocaproate during starvation conditions in the muscle cell?
Leucine is a strictly ketogenic amino acid. As such, its carbon skeleton (alpha-ketoisocaproate) produced by branched chain aminotransferase will be broken down into acetyl-CoA, and then completely oxidized (on site) to CO2 + H2O by the TCA cycle.
Note: The muscle cell cannot synthesize ketone bodies!
The alpha-ketoglutarate concentration remains relatively constant in the muscle cell. Why does alpha-ketoglutarate NOT accumulate NOR deplete under starvation conditions? A diagram linking aminotransferase reactions may help in your explanation.
Alpha-ketoglutarate is produced by Alanine aminotransferase for every N atom moved from Glutamate to Alanine (subsequentely exported from muscle to liver). This alpha-ketoglutarate is simply cycling with other ‘upstream’ aminotransferases (e.g., TAT, branched chain aminotransferase, AST, etc.) that all require alpha-ketoglutarate as substrate. Alpha-ketoglutarate is a transport mechanism for N from upstream amino acids to the N-acceptor, pyruvate. As the N is 1:1, there is no net change in alpha-ketoglutarate level. No depletion. No accumulation.
What is the fate of the nitrogen that is moved between metabolites by aminotransferases during starvation?
In a working muscle, the alpha-amino group nitrogen is moved from the respective amino acid (breakdown pathways) to primarily pyruvate, forming alanine. Alanine leaves the muscle cell (alanine transporter), enters the blood and is imported by the liver (alanine transporter). The liver further metabolises the nitrogen in Alanine to eventually produce urea. To a lesser extent, some of the nitrogen will also be used to make Glutamine (Glu + NH4+ > Gln). Glutamine is transported to the liver to help move the N and produce urea.
What is the function of the blood protein albumin?
Albumin is a protein that binds fatty acids in the blood (a fatty acid carrier protein). Its function is to transport fatty acids from adipose tissue to the cells that need them (e.g., muscle cell). This occurs during fasting or starvation.
The following table shows the fatty acid concentration in blood in a healthy human. Please explain and rationalize the data.
Fatty acids are >1000x more likely to be bound to albumin than exist free in blood! Fatty acids are primarily hydrophobic (long hydrocarbon tail) and have minimal hydrophilic character (carboxylic head group). As such they have low stability in aqueous environments such as blood. This is observed experimentally by the low concentration of free FA in blood (note: nM range). In order to mobilize large amounts of FA from adipose tissue to cells that need them, a transporter protein such as albumin is required. The majority of FA in blood are thus bound to albumin, effectively raising the stability up to ~30,000 fold!
Albumin has 7 fatty acid-binding sites as determined by X-ray crystallographic studies. During normal ‘well-fed’ conditions, albumin binds with approximately 0.1-2mol fatty acid per mol albumin. During fasting or severe starvation conditions the fatty acid/albumin molar ratio increases 6-7. Discuss and rationalize these findings.
After eating (times of plenty, fed part of fed-fast cycle, insulin-signaling), most cells in the body use glucose as a fuel source. Fatty acids coming from the intestinal tract will be esterified to glycerol and carried as triacylglycerols in chylomicrons. FAs are not picked up by albumin from the intestine. Thus, most albumin molecules will have little FA bound to them (0-2 FA per albumin, corresponding to the lower 50uM range). However, upon fasting, glucagon-signalling causes the mobilization of fat stores in adipocytes. FAs signalling causes the mobilization of fat stores in adipocytes. FAS are released into the blood where they are bound by albumin. Now the albumin molecules become heavily loaded: ~6-7 FA per albumin!
Pyridoxal phosphate
Insulin is given intravenously (i.e., injected into the blood) in the treatment of diabetes. Why can this hormone, a small protein, NOT be taken orally? [2]
The biological activity of insulin would be destroyed by the low pH of the gastric juices in the stomach as well as by the proteases (e.g., pepsin, chymotrypsin, etc.) that act in the stomach and small intestines.
Even if insulin escaped degradation and refolded to its active state, it would NOT enter the blood from the intestine. The transporters that line the intestinal lumen transport free amino acids, NOT intact proteins.
Some microorganisms in the Archaea evolutionary branch thrive in extreme environments of high temperature and high pH. Archael membrane lipids are unique and consist of the compound shown.
How could a biological membrane be constructed with this lipid in the archaea?
The hydrocarbon chain would form the hydrophilic core and span the membrane (analagous to 2x acyl chains of e.g., eukaryotic membranes). The free hydroxyl groups (or polar derivatives) on either side would form polar ‘head groups’ analogous to polar heads of 2x leaflets of eukaryotic membranes.
Some microorganisms in the Archaea evolutionary branch thrive in extreme environments of high temperature and high pH. Archael membrane lipids are unique and consist of the compound shown.
Provide 3 differences of the archael membrane lipid compared to glycerophospholipids found in the evolutionary branches of bacteria and animals we studied in class. Explain.
- Archaea lipids have glycerol linked to hydrocarbon chains by ether linkages. Glycerophospholipids have glycerol linked to hydrocarbon chains by ester linkages.
- The hydrocarbon chains of archaea lipids (C32) are 2x as long as that of glycerophospholipids (C16-20)
- The hydrocarbon chain of archaea lipid is branched with methyl groups (fully saturated isoprenoid units), whereas, hydrocarbon chains of phospholipids are NOT branched (e.g., palmitate).
Some microorganisms in the Archaea evolutionary branch thrive in extreme environments of high temperature and high pH. Archael membrane lipids are unique and consist of the compound shown.
Give a reason why the archaeal membrane lipid provides thermal OR chemical stability to the membrane? Briefly justify your answer.
Thermal: The archaea compound is stable at high temperatures because of the many van der waals interactions down the carbon chain. There is also limited mobility as there is no bilayer in this membrane as the ‘leaflets’ are covalently linked. The methyl groups would prevent further rotational mobility and increase van der waals interactions. All these factors increase TM of membranes.
Chemical: The ether linkages are stable to chemical hydrolysis. For example at pH 10, ester linkage would hydrolyze with OH nucelophilic attack. Lipases cannot hydrolyze.
Plargonate is a 9-carbon saturated fatty acid found in plants and many fruits that we commonly eat. What is the ATP yield of pelargonate when it is completely oxidized to CO2 and H2O? What is the net H2O cost or H2O generated when pelargonate is completely oxidized to CO2 and H2O?
How is the catabolism of strictly ketogenic amino acids and even-numbered carbon fatty acids similar?
Both ketogenic amino acids and even-numbered carbon FAs will only produce acetyl-CoA, which can be directly oxidized by the TCA cycle and oxidative phosphorylation to produce ATP, OR it can be used to make ketone bodies in the liver and then exported (e.g., to the brain) again for ATP production.
How is the catabolism of glucogenic amino acids and odd-numbered fatty acids similar?
Both glucogenic amino acids and odd-numbered carbon FAs can lead to the generation of acetyl-CoA AND TCA cycle intermediates that can subsequently be used as precursors to build glucose (gluconeogenesis). Thus, they can both be used as a carbon source to fuel the brain by generating glucose.
Suppose your diet was completely derived from strictly ketogenic amino acids and even-numbered carbon fatty acids. What do you expect to your muscle tissue? Why?
Muscle tissue will waste away. The body has no direct supply of glucose, so will need to make glucose. Once glycogen stores run out (~24hrs), gluconeogenesis will have to supply blood glucose. Neither strictly ketogenic amino acids, nor even-numbered carbon FAs can be used as a carbon source in gluconeogenesis. The body will thus break down muscle tissue (primarily skeletal muscles) as a source of gluconeogenic amino acids. These can be converted to glucose to continue to supply the brain with its required fuel source.
Each round of fatty acid synthesis uses 4 general types of chemical reactions. List these 4 types of chemical reactions in the proper order that they will occur during each round of FA synthesis.
- Condensation
- Reduction
- Dehydration
- Reduction
The synthesis of palmitate by fatty acid synthase (FAS) produces 6H2O molecules. Why are only 6 water molecules produced when there are a total of 7 dehydration reactions during FA synthesis?
The synthesis of palmitate requires 7 rounds of synthesis, including seven dehydration reactions (ß-hydroxyACP dehydratase). After the last round of synthesis, the 16-carbon acyl chain is still attached by thioester linkage to ACP. Thioesterase uses 1 H2O (as substrate) to hydrolyze palmitoyl-ACP to release palmitate. (7-1 = 6)
Why does a mammal go to all the trouble of making urea from ammonia rather than simply excreting ammonia as many fish do?
When fish release ammonia into the surrounding aqueous environment, it is diluted to non-toxic levels. The ammonia produced (e.g., by amino acid catabolism) in mammals cannot be sufficiently diluted in the tissues and blood to avoid accumulating toxic amounts. Urea is much less toxic than ammonia. Ammonia is also highly toxic to fish, but its dilution into the aquatic environment results in concentrations that are less than toxic.
Arctic animals would be expected to have a higher cholesterol content in the cell membranes in their extremities because:
Cholesterol’s steroid nucleus prevents close packing of long-chain fatty acids in adjacent lipids, increasing membrane fluidity at low temperatures.
Which of the following molecules are NOT essential for a healthy human adult? (i.e., these molecules can be synthesized de novo)
a. carbamate
b. linoleate
c. leucine
d. biotin
e. pyridoxine
f. ornithine
g. Two of the above
h. None of the above
g. Two of the above
The degradation of arachidonate in the mitochondrial matrix requires X rounds of breakdown and produces Y molecules of acetyl-CoA.
The degradation of arachidonate (C20:4 all cis Δ5,8,11,14) in the mitochondrial matrix requires 9 rounds of breakdown and produces 10 molecules of acetyl-CoA.
The human genetic disease phenylketonuria (PKU) can result from:
Inability to regenerate tetrahydrobiopterin (THB) from dihydrobiopterin
Each round of fatty acid breakdown uses 4 general types of chemical reactions. The correct order that these chemical reactions proceed for each round of fatty acid breakdown are:
- Oxidation
- Hydration
- Oxidation
- Thiolysis
What are the substrates/products required to complete the reaction catalyzed by glutamate dehydrogenase?
What is the enzymatic process performed by this enzyme?
Note: No cofactor necessary for this enzyme.
Glutamate dehydrogenase performs oxidative deamination.
Prove that this is or is not an oxidation-reduction (redox) reaction.
This is a redox reaction. The carbon substrate (Glu) is oxidized as there is a loss of electrons going to the product (alpha-KG). Both H2O and NH4+ have four e- pairs and are not involved in the redox reaction. (i.e., the first step in the reaction with NAD(P)+ to form the reaction intermediate is a redox reaction).
Briefly describe how the hormone glucagon helps control fat catabolism.
Glucagon signals adipocytes and via the cAMP-dependent signal transduction cascade activates protein kinase A (pkA). PKA phosphorylates hormone sensitive lipase (and long with CPI) allows TAG molecules to be broken down into fatty acids and glycerol. These are then released into the blood. Tissues that catabolize FAs will bring them into the cytosol, link them to CoASH and via the carnitine shuttle move them into the mitochondrial matrix, to perform oxidation. For cells that have acetyl-CoA carboxylase (ACC), glucagon also ensures that ACC is phosphorylated and inactive. This ensures that CATI and the carnitine shuttle are working.
Compare the last reaction of ß-oxidation of an even-numbered carbon fatty acid to the first step of cholesterol synthesis.
Name a tissue type that each of these metabolic processes operate in.
Specify the cellular compartments that each of these two metabolic processes operate in within the tissue.
Both reactions are performed by thiolases (aka acyl-CoA acetyl transferase). ß-oxidation uses coenzyme A to cleave acetoacetyl-CoA into 2x acetyl-CoA. Thiolase of ß-oxidation operates in the mitochondrial matrix of ALL cells except the brain (e.g., epithelial cells).
In cholesterol synthesis, this reaction operates in ‘reverse’: i.e., two acetyl-CoA combine to form acetoacetyl-CoA with the release of CoASH. Thiolase of cholesterol synthesis operates in the cytoplasm the cells of the liver (or intestine).
List five general ways metabolic pathways are regulated. Briefly describe each. Do NOT provide specific examples.
- Transcriptional regulation of the gene that expresses the rate-limiting enzyme thereby changing [E].
- Translational regulation of the mRNA that expresses the rate-limiting enzyme thereby changing [E].
- Reversible phosphorylation of the rate-limiting enzyme. Phosphorylation could increase or decrease the enzyme activity.
- Control the enzyme degradation rate by changing the half-life (Ub-dependent degradation)
- Allosteric activators or inhibitors of the rate limiting enzyme will increase or decrease the enzyme activity, respectively.
Taking out the trash costs money! Damaged or misfolded proteins provide no service to a cell and thus have to be discarded. Specific E3s (ubiquitin ligases) recognize by binding these damaged and unfolded proteins. As we learnt in class, these recognized proteins are subsequently hydrolyzed into their constituent amino acids. Breaking peptide bonds releases energy, yet there is a substantial net energy cost to remove these non-functional proteins. Explain why. Be specific. Try to quantitate your explanation for full marks.
- Proteins removed by the Ub-dependent pathway have to be at minimum tetra-ubiquitinated. Attachment of 1 (one) Ub (by E1, E2, E3) costs the equivalent of 2 ATP. Thus at least 8 ATP are required.
- Once ubiquitinated, the target protein is degraded by the 26S proteasome. Unfolding the target protein by the 19S cap, as well as threading the protein into the core are energy costing or ATP-dependent processes. Presumably the longer the polypeptide the higher the ATP cost (a stoichiometric equivalent here is unknown).
- Other energy considerations are the cost of protein synthesis of the protein degradation machinery, (i.e., E1, E2, E3 suite of enzymes, Ub, 26S proteasome, and the suite of cellular peptidases).
Draw the reaction catalyzed by the KR domain of mammalian FAS during the 5th round of synthesis. Include names and structures of substrates and products at pH 7 (generic names will suffice). Balance all atoms, including H atoms. Also include any required cofactors.
Compare the reaction catalyzed by the KR domain of mammalian FAS to the KR reaction catalyzed in bacteria. In your answer provide an advantage of the mammalian catalyzed reaction.
The ß-ketoacyl reductase reaction in both mammals and bacteria occurs in the cytosol. The mammalian KR domain is part of FAS. The carbon substrate and product are attached to ACP, another domain of FAS. Thus the carbon produt of KR (pathway intermediate) remains tethered to FAS and rapidly diffuses to the subsequent active site (i.e., DH domain of FAS).
In the KR reaction catalyzed in bacteria, the reaction is performed by the KR enzyme, a free enzyme. The carbon product of KR (pathway intermediate) diffuses into the cytoplasm and has to diffuse into the active site of the free DH enzyme. This is not as efficient as the mammalian catalyzed reaction. Tethering the pathway intermediates and having all enzymes joined as domains is much more efficient to ‘flux’ a metabolite through a pathway.
Name all the enzymes required to allow protein hydrolysis to occur in reaction step 2.
Ubiquitin activating enzyme (UbA), conjugating enzymes (UbC), and ligases (UbL). Also 26S proteasome and cellular peptidases.
Write out a net reaction for the biosynthesis of L-carnitine starting with N6-trimethyllysine (rxns 3-6).
Although humans have a biosynthetic pathway for carnitine (rxns steps 3-6), why can humans actually NOT make carnitine de novo?
The biosynthetic pathway depends on both Lysine and Methionine which are both essential amino acids! These must be supplied from the diet.
Provide a diagram that shows the function of the carnitine shuttle. Include all necessary proteins and label the cellular compartments. Clearly indicate the flow of the required cofactors and fatty acyl groups.
In addition to arginine dietary supplementation, patients with argininosuccinase deficiency also need to be supplemented with oxaloacetate. Suggest why both arginine and oxaloacetate are important.
These patients do NOT have argininosuccinase activity and thus their urea cycle does NOT turn. They cannot form urea and do NOT have the ability to eliminate excess nitrogen. Arginine allows them to regenerate ornithine allowing for N incorporation from peripheral amino acid catabolism. The ornithine incorporates NH4+ and Aspartate to synthesize argininosuccinate, which is secreted and excreted by the kidney. This proess however also uses up oxaloacetate (OAA) in the matrix for every argininosuccinate excreted. To prevent this loss of OAA, the diet is supplemented with OAA.
Imagine a hypothetical situation where synthesis and breakdown of the fatty acid stearate occured at the same time.
How is such a futile cycle prevented in an epithelial cell? What about in a liver cell?
A peripheral cell (e.g., epithelial cell) does not have the enzymes (ACC, FAS) to make fatty acids, and as such cannot perform such a futile cycle. Only the liver, adipocytes, and mammary gland cells perform FA synthesis.
In a liver cell, ACC is either phosphorylated (inactive) or dephosphorylated (active). This reversible phosphorylation is controlled by hormones (glucagon phoshorylates, insulin dephosphorylates), or other cellular mediators (e.g., AMP dependent kinase).
When ACC is active, its product malonyl-CoA is made resulting in increased [malonyl-CoA]. This results in FA synthesis (substrate for FAs) and negative allosteric regulation of CATI, preventing fatty acyl transport into the matrix, thereby depriving the ß-oxidation enzymes of their substrates.
When ACC is inactive, there is no malonyl-CoA and therefore no substrate for FA synthesis. In this case there is no inhibition of CATI and FAs are transported by the carnitine shuttle into the matrix for oxidation.
Imagine a hypothetical situation where synthesis and breakdown of the fatty acid stearate occured at the same time.
What would be the ATP cost or yield for a cell performing such a futile cycle?
Final answer (either accepted)
Costs 27 ATP
Costs 36 ATP
Why are triacylglycerols (aka triglycerides) the main energy form in animals? [2]
- FA are almost completely reduced (little oxygen) > can harvest more energy per gram (38kJ/g) for FAs vs 17kJ/g for carbs)
- TAG are hydrophobic and don’t need water to store compared to glycogen. Thus, we can pack more TAG in a given volume.
Briefly describe the structure of the 7 major types of lipids.
1. Fatty acids (FA) - carboxylic acids containing long hydrocarbon chains that can be saturated or unsaturated.
2. Triacylglycerols - glycerol that has FA esterified to each of the OH groups.
3. Glycerophospholipids - composed of a glycerol backbone to which 2 FAs are attached, but carbon #3 is linked by a phosphodiester bond to phosphate, which in turn is linked to a head group (alcohol)
4. Sphingolipids - have sphingosine as a base that consist of a polar head group attached by -OH, and also have a free -OH group and one FA chain attached by amide linkage
5. Steroids - lipids containing core of 4 fused rings (A-D): A-C 6C, D 5C. Steroid + alcohol = sterol. Planar, rigid and sterols are amphipathic. See figure.
6. Waxes - esters of FA linked to long chain alcohols - highly hydrophobic and non-polar (e.g., plant leaves)
7. Eicosanoid - signaling molecules involved in the inflammatory response.
Describe biological membranes:
Composition, structure, permeability
- Sheet like structures composed of lipids and proteins and usually 2 bilayers.
- Form closed boundaries between cells and compartments within cells.
- 3 major lipid components: glycerophospholipids, sphingolipids, and sterols (in animals cholesterol, plants use other sterols as well) Note: Bacteria do not have biosynthetic pathways for sterol, but may incorporate some exogenous sterols into their membranes.
- Basic structure: bilayer (inner and outer leaflets) and small polar (glucose) or charged (ions) molecules.
- Permeable to small molecules (O2 and CO2) and small hydrophobic molecules (steroid hormones)
Describe the fluid mosaic model of membranes.
- Fluid lipid bilayer with proteins floating in it.
- Proteins and lipids can freely diffuse laterally, but flip flops are rare
- Membranes have appropriate fluidity for performing biological functions for a given temperature.
Describe Lo and Ld.
Liquid-ordered state (Lo): When T is below the appropriate temperature for optimal membrane fluidity (Tm), phase transition occurs and lipids become semi-solid or gel-like.
Liquid-disordered state (Ld): When T is above the appropriate temperature for optimal membrane fluidity (T>Tm), lipids become more disordered and the membrane becomes too fluid.
How do organisms regulate membrane fluidity? [3]
1+2: Changing the FA attached to phospholipids: shorter FA > lower Tm, greater degree of saturation > higher Tm.
- Animals control fluidity by using cholesterol to adjust the orderered packing of FA acyl chains.
Describe how cholesterol affects membrane fluidity.
Cholesterol broadens the range of appropriate membrane fluidity.
Below Tm - cholesterol prevents tight packing of acyl chains > slows transition to semi-solid state
Above Tm - cholesterol prevents rotation of acyl chains > enhances tight packing of acyl chains > slows transition to fluid-like state
Describe the preparatory step of FA synthesis.
Acetyl-CoA carboxylase (ACC) initiates FA synthesis by converting acetyl-CoA to malonyl-CoA.
- ACC adds CO2 to acetyl-CoA generating ‘activated acetyl-CoA’ (=malonyl-CoA).
- 1 molecule of ATP is hydrolyzed in the process.
- ACC has biotin (vitamin B7) as a prosthetic group attached to the Lysine residue of ACC.
- Occurs in 2 steps:
1. ACC-biotin + ATP + HCO3- > ACC-biotin-COO- + ADP + Pi + H+
2. ACC-biotin-COO- + acetyl-CoA > malonyl-CoA + ACC-biotin (irreversible, committed step)
Note: In a cell there is always CO2 + H2O ⇌ HCO3- + H+
Does FA synthesis require both acetyl-CoA and malonyl-CoA?
Initiation of FA requires both acetyl-CoA and malonyl-CoA, whereas elongation requires only malonyl-CoA.
Describe the structure of Fatty Acid Synthase and list its 7 domains.
FAS consists of a single polypeptide chain with multiple enzymatic activities and 7 functional domains. Works as a homodimer.
Note: In bacteria these are 7 individual proteins and not a single complex.
7 domains: KS-MAT-DH-ER-KR-ACP-TE
What are the full names of each functional domain of mammalian fatty acid synthases?
KS: ß-ketoacyl-ACP synthase
MAT: Malonyl/acetyl-CoA-ACP transferase
DH: ß-hydroxyacyl-ACP dehydratase
ER: Enoyl-ACP reductase
KR: ß-ketoacyl-ACP reductase
ACP: acyl-carrier protein
TE: Thioesterase
What are the two thiol groups found in FAS in mammals? Describe their significance.
- Acyl carrier protein (ACP) contains a long flexible phosphopantetheine group (similar to CoASH, dervied from vitamin B5). It can bind acyl groups via reactive thiol and the long flexible arm allows acyl-groups to be moved around from one enzymatic activity to the next. All acetyl or malonyl groups are first loaded onto ACP.
- Reactive Cysteine residue in the ß-ketoacyl ACP synthase (KS) domain can also bind acyl groups.
Describe how fatty acid synthase is charged.
- MAT transfers acetyl group from acetyl-CoA to ACP. ACP swings to KS domain where acetyl group is transferred from thiol group of ACP to thiol group of Cys in KS domain. This only happens in round 1.
- MAT transfers malonyl group from malonyl-CoA to ACP and ACP swings next to KS domain. This happens every round (only difference is length of acyl group attached to KS domain)
Describe the FAS condensation reaction.
KS transfers an acetyl group only malonyl group while carboxyl group leaves as CO2 (same carbon that was added by ACC in activation step prior).
As a result ß-ketoacyl-ACP (e.g., in round 1 acetoacetyl-ACP) is formed.
Why does ACC add CO2 to an acetyl group?
The addition of CO2 made the acetyl group more reactive and CO2 is a good leaving group. This drives the reaction forward since the reaction is irreversible.
Does FAS require ATP?
Not directly, but it is used by ACC in the preparatory step.
Describe the first FAS reduction reaction.
ACP arm loaded with ß-ketoacyl group swings to KR domain (ß-ketoacyl ACP reductase), where ß-ketocarbon is reduced to a hydroxyl group, forming D-ß-hydroxyacyl-ACP.
NADPH serves as an electron donor and is converted to NADP+
Describe the FAS dehydration reaction.
ACP arm with D-ß-hydroxyacyl moves to DH domain (D-ß-hydroxyacyl ACP dehydratase). DH removes H2O (OH from ß-carbon and H from α-carbon) forming trans-Δ2-enoyl-ACP.
ß-hydroxybutyryl-ACP
trans-Δ2-Butenoyl-ACP
Butyryl-ACP
ß-ketobutyryl-ACP
Describe the second FAS reduction reaction.
ACP arm loaded with trans-Δ2-enoyl group swings to ER domain (enoyl-ACP reductase). ER reduces the double bond forming acyl-ACP. NADPH is used as a source of electrons and is oxidized to NADP+.
What are the enzymes involved in fatty acid synthesis (excluding the preparatory and charging steps)?
How does FAS prepare for another round of synthesis cycle and acyl formation?
Transfer of the acyl chain from ACP to KS. ACP swings to KS domain and KS transfers acyl group from ACP to Cys in KS domain. The cycle then continues.
Note: The original acetyl group is at the end of the growing chain. The chain grows from the carbonyl end.
What is the final product of fatty acid synthesis in mammals?
How many rounds of FAS does it require?
Palmitoyl-ACP is the final product: requires 7 rounds.
Then the TE domain (thioesterase) hydrolyzes the C16 acyl chain from ACP with water (hydrolysis) yielding palmitate.
What is the stoichiometry of FA synthesis for palmitate synthesis?
Give the stoichiometry for FAS, ACC, and overall.
FAS (7 rounds)
1 acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 20 H+ > palmitate + 7CO2 + 14 NADP+ + 6H2O + 8 CoASH
Note: 7 H2O produced, but one is used by thioesterase (TE)
ACC (7 rxns)
7 acetyl-CoA + 7 CO2 + 7ATP + 7H2O > 7 malonyl-CoA + 7ADP + 7Pi + H+
Overall
8 acetyl-CoA + 7ATP + 14NADPH + 6H+ + H2O > palmitate (C16:0) + 14NADP+ + 8CoASH + 7ADP + 7Pi
What happens to palmitate after synthesis? [3]
It can be elongated, desaturated, and branched by separate enzymes.
- Palmitate can be desaturated by desaturases that will introduce double bonds using O2 and NAD+ (or NADP+) to generate cis double bonds. There are several desaturases introducing double bonds at different locations. Mammals cannot double bond beyond C9, so need to obtain from dietary sources.
- Palmitate can be converted to longer FAs by set of enzymes called elongases.
- There are also branching enzymes that can be synthesized from branched FAs from palmitate (some bacteria, sea lions).
Mammals can introduce double bonds beyond C9 in fatty acid desaturation, so there is no need to obtain them from dietary sources.
True or false?
False.
Mammals cannot induce double bonds beyond C9, so there is a need to obtain them from dietary sources.
Where is acetyl-CoA generated?
Acetyl-CoA is generated by carbohydrate, protein, and FA catabolism in the mitochondrial matrix, but FA synthesis occurs in the cytosol.
How is acetyl-CoA shuttled to the cytosol for FA synthesis?
What 8 enzymes are involved?
Acetyl-CoA is shuttled to the cytosol in the form of citrate (produced in TCA cycle from acetyl-CoA and oxaloacetate by citrate synthase) via citrate transporter.
Once in the cytosol citrate lyase uses ATP and converts citrate back to acetyl-CoA and oxaloacetate.
Oxaloacetate needs to be transported back to the mitochondria to prevent depletion of the TCA cycle. First oxaloacetate is converted to malate by malate dehydrogenase (uses NADH). Malate can be directly transported back to mitochondria via malate-ketoglutarate transporter and then converted back to oxaloacetate in the matrix.
However, majority of malate is converted to pyruvate by malic enzyme producing NADPH (used in FA synthesis). Pyruvate then enters the mitochondria via pyruvate transporter where it is converted to oxaloacetate by pyruvate carboxylase.
Note: NADPH is also produced by pentose phosphate pathway.
Malate
Malate
Citrate
Citrate
Citrate
From left to right:
Leucine, Isoleucine, Valine
Valine
Isoleucine
Methionine
Proline
Methionine
Tryptophan
Tryptophan
What is the net ATP yield for complete oxidation of acetyl-CoA to CO2 and H2O?
10 ATP
Biotin (B7)
Pyridoxine (B6)
Tetrahydrobiopterin
Tetrahydrobiopterin
Protein secretory pathway mutants in mice. Experiments are carried out in mice on low cholesterol diet to further gain insight to the transcriptional regulation of cholesterol metabolism. Four (4) mutant mice are made that they have protein secretory pathway effects. The first mutant does not express the serine protease (SP-), i.e. the serine protease is NOT present. The second mutant expresses the serine protease but only in the endoplasmic reticulum (SPER). The third mutant expresses the metalloprotease but only to the endoplasmic reticulum (MPER). The fourth mutant expresses both the serine protease and metalloprotease, but only to the ER (SPER/MPER). HMG-CoA reductase levels were measured in the wild type and mutant mouse strains to see the affect of the mutations. The results are shown.
What does the lack of HMG-CoA expression in the SP- mouse strain tell you about the transcription regulation of cholesterol metabolism?
Lack of the serine protease prevents release of the transcription factor from SREBP that normally turns on HMG-CoA reductase transcription leading to HMG-CoA reductase enzyme expression. Although the metalloprotease is present, it alone cannot release the transcription factor domain. This 2nd cleavage by metalloprotease is somehow dependent on the serine protease performing the 1st cleavage on SREBP. Possible steric hindrance or a conformational change in the substrate.
Protein secretory pathway mutants in mice. Experiments are carried out in mice on low cholesterol diet to further gain insight to the transcriptional regulation of cholesterol metabolism. Four (4) mutant mice are made that they have protein secretory pathway effects. The first mutant does not express the serine protease (SP-), i.e. the serine protease is NOT present. The second mutant expresses the serine protease but only in the endoplasmic reticulum (SPER). The third mutant expresses the metalloprotease but only to the endoplasmic reticulum (MPER). The fourth mutant expresses both the serine protease and metalloprotease, but only to the ER (SPER/MPER). HMG-CoA reductase levels were measured in the wild type and mutant mouse strains to see the affect of the mutations. The results are shown.
What does the normal HMG-CoA expression level in the SPER mouse strain tell you about the transcription regulation of cholesterol metabolism?
The serine protease is normally in the Golgi. If serine protease is expressed in the ER, the SREBP will be cleaved already in the ER by the serine protease. Once the cleaved SREBP moves to the Golgi (due to low [cholesterol] signalling), the metalloprotease can perform the 2nd cleavage. This is just like the wild type except the serine protease has pre-cleaved SREBP in cholesterol-independent fashion.
Protein secretory pathway mutants in mice. Experiments are carried out in mice on low cholesterol diet to further gain insight to the transcriptional regulation of cholesterol metabolism. Four (4) mutant mice are made that they have protein secretory pathway effects. The first mutant does not express the serine protease (SP-), i.e. the serine protease is NOT present. The second mutant expresses the serine protease but only in the endoplasmic reticulum (SPER). The third mutant expresses the metalloprotease but only to the endoplasmic reticulum (MPER). The fourth mutant expresses both the serine protease and metalloprotease, but only to the ER (SPER/MPER). HMG-CoA reductase levels were measured in the wild type and mutant mouse strains to see the affect of the mutations. The results are shown.
What does the lack of HMG-CoA expression level in the MPER mouse strain tell you about the transcription regulation of cholesterol metabolism?
The metalloprotease is normally found in the Golgi, not the ER. The metalloprotease cannot cleave the SREBP without prior cleavage by serine protease. SREBP will not be cleaved by the metalloprotease in the ER, as the serine protease is still residing normally in the Golgi. When SREBP moves to the Golgi, there will be cleavage by the resident serine protease, but no release of the transcription factor domain as there is no metalloprotease present.
Protein secretory pathway mutants in mice. Experiments are carried out in mice on low cholesterol diet to further gain insight to the transcriptional regulation of cholesterol metabolism. Four (4) mutant mice are made that they have protein secretory pathway effects. The first mutant does not express the serine protease (SP-), i.e. the serine protease is NOT present. The second mutant expresses the serine protease but only in the endoplasmic reticulum (SPER). The third mutant expresses the metalloprotease but only to the endoplasmic reticulum (MPER). The fourth mutant expresses both the serine protease and metalloprotease, but only to the ER (SPER/MPER). HMG-CoA reductase levels were measured in the wild type and mutant mouse strains to see the affect of the mutations. The results are shown.
What does the overexpression level of HMG-CoA in the SPER/MPER double mutant mouse strain tell you about the transcription regulation of cholesterol metabolism?
Both metalloprotease and serine protease are expressed in the ER. The substrate SREBP also resides there regardless of cholesterol levels. Thus, serine protease (1st) and metalloprotease (2nd) will cleave SREBP releasing the transcription factor into the cytoplasm where it will in turn enter the nucleas and turn ON HMG-CoA reductase gene transcription. High amounts of HMG-CoA reductase are expressed independent of cholesterol concentration.
What is the nature of the linkage formed in creation of an internal aldimine in an aminotransferase?
Schiff Base
What is the reaction carried out by the thioesterase (TE) domain of Fatty Acid Synthase?
Hydrolysis
What is the NET charge of phoshphatidylserine at pH 7.4 (i.e., physiological conditions)?
- 1 phosphate
- 1 COO-
+1 NH3+
NET: -1
Name two enzymes that utilize ATP and produce AMP and pyrophosphate.
Argininosuccinate synthetase (ASS)
Acyl CoA synthetase (ACS)
What enzyme catalyzes the most important route for the incorporation of free ammonia/ammonium ions into amino acid (assume normal ammonia levels in the blood)?
Glutamine synthetase
What enhances the activities of both Acetyl CoA Carboxylase and HMG-CoA Reductase?
Dephosphorylation
An insufficient amount of this vitamin will result in decreased synthesis of malonyl-CoA leading to decreased fatty acid synthesis. What vitamin is this?
Biotin (B7)
Odd numbered fatty acids are directly broken down to acetyl-CoA and what other molecule?
Odd numbered fatty acids are directly broken down into acetyl-CoA and propionyl-CoA (which is in turn converted to succinyl-CoA and enters the TCA cycle)
Some lipases are activated by hormones.
True or false?
True
Some lipases are activated by proteins which directly bind to them
True or false?
True.
Some lipases function near the lipid droplet-cytoplasm interface of adipocytes.
True or false?
True.
All lipases require water as part of their catalytic mechanism of action.
True or false?
True
All lipases produce three fatty acids and glycerol.
True or false?
False.
Regarding this molecule, which statement is correct:
a. It is derived from cholesterol and is extremely hydrophobic
b. It is derived from acetyl-CoA and contains 5 isoprenoid units
c. It is an activated form of cholesterol and is produced by condensation of two geranyl pyrophosphate units
d. It is a cholesterol synthesis pathway intermediate and contains 6 isoprenoid units
e. It is a keton body produced from 5 HMG-CoA units
d. This molecule is squalene. It is a cholesterol synthesis pathway intermediate and contains 6 isoprenoid units.
Which of the following is an omega-6 fatty acid?
a. arachidonic acid
b. Hexanoic acid
c. trans oleic acid
d. stearic acid
e. C18:2 (cis Δ9,12) carnitine (linoleoyl carnitine)
d. Two of the above
a. arachidonic acid
Consider the enzyme mechanism for the aminotransferase AST. Which of the following is INCORRECT, when the enzyme encounters relatively high concentrations of Asp and has sufficient alpha-ketoglutarate for activity?
a. AST has a bimolecular ping-pong mechanism
b. Asp and alpha-ketoglutarate at the active site are required for the formation of pyridoxine phosphate (PMP).
c. The mechanism has 4 initial steps that allow oxaloacetate release, followed by the reverse of these steps in the transfer of amino group to alpha-ketoglutarate with Glu formation.
d. If the Asp substrate is radioactively labelled with 15N, it follows that any PMP formed should also be radioactive.
e. If the Asp substrate is radioactively labelled with 15N, it follows that the Glu product of the reaction will also be radioactively labelled.
b. Asp and alpha-ketoglutarate at the active site are required for the formation of pyridoxamine phosphate (PMP).
All of the following are phosphate-containing lipids found in biological membranes EXCEPT for one. Indicate the exception.
Phosphatidylserine
Cardiolipid
Farnesyl pyrophosphate
Sphingomyelin
Phosphatidylinositol
Farnesyl pyrophosphate
During the complete oxidation of a fatty acid (e.g., performed by a bear in hibernation) electrons in the fatty acid are removed and ultimately end up on what molecule?
H2O
What is the ATP yield if the structure below is completely oxidized to CO2 and H2O?
90.5
TAGs and cholesterol synthesized in the liver can be transported to other cells of the body through the help of…
Lipoprotein particles
Note: albumin transports FAs not TAGs nor cholesterol
Write the balanced equation for the transamination of phenylalanine to phenylpyruvate, and indicate the structures of the reactants and products at pH 7.
Identify any cofactors and a possible enzyme needed to accomplish the reaction.
Cofactors: pyridoxal phosphate
Enzymes: likely alanine aminotransferase or another aminotransferase
The cellular pool of NAD+ is limited. Show schematically (i.e., use a diagram) how NAD+ is recycled in aerobic systems during ß-oxidation. Note any enzyme or enzyme complexes required.
Where is the labelled carbon found (14C) when the following molecules are added to a liver homogenate carrying out stearate (C18:0) synthesis?
a. H14CO3-
b. see image
A pure phospholipid bilayer is composed of an equal mixture of palmitoylsphingomyelin and dipalmitoylphosphatidylserine (50:50). Two other membranes are made. Membrane A is composed of an equal mixture of oleoylsphingomyelin and dioleoylphosphatidylserine (50:50). Membrane B is identical to membrane A but also contains 20% cholesterol. Draw the phase transition diagrams of all three membranes. Clearly label the curves.
If 15N-labelled aspartate is fed to animals, many amino acids labelled with 15N quickly appear. Thoroughly explain this observation. Follow the nitrogen!
The reactions catalyzed by aminotransferases in the liver are reversible, and many aminotransferases use Glutamate as the alpha-amino group donor. After 15N-labelled Glutamate is formed by aspartate aminotransferase (AST), the 15N atom will be quickly distributed among the other amino acids that are products of other aminotransferases
Lysophosphatidylcholine (LPC) has the same structure as phosphatidylcholine except that one of the ester-linked fatty acyl chains has been removed by hydrolysis. Draw the structure of LPC: 2-stearoyl-phosphatidylchonline at pH 7.
When a small amount of the lysophosphatidylcholine (LPC): 2-stearoyl-phosphatidylcholine is added to water, the LPC molecules enter the solution as a monomeric species. As more LPC is added, a concentration is reached (the critical micelle concentration) at which the monomers associate to form micelles. The critical micelle concentration of LPC is 0.4uM. The micelles have an average particle weight (the sum of molecular weights of constituent monomers) of 149,340. The molecular weight of 2-stearoyl-phosphatidylcholine is 524 g/mol.
Sketch the LPS structure found at an LPS concentratino of 0.5uM.
Calculate the number of 2-stearoyl-phosphatidylcholine molecules in the average micelle.
Unlike LPC, indicate why diastearoylphosphatidylcholine (PC) with two acyl chains is more likely to form another lipid aggregate structure. What structure is this?
- One micelle = 149,340 g/mol and comprised of LPC = 524 g/mol. Therefore 149,340/524 = 285 LPC molecules in one micelle.
- LPC has only 1 tail and 1 large polar head group, which has a cone shape which favours micelle formation.
- PC has 2 tails (both saturated) and will have a cyllinder shape. This favours bilayer structures such as biological membranes or liposomes or vesicles.
Which structure is cholesterol?
Structure E.
Malonyl-CoA, labelled with radioactive 12C at the carbonyl carbon is used in addition to unlabelled acetyl-CoA and other substrates required by FAS I complex to make palmitate (C16:0). The enzyme acetyl CoA carboxylase is absent from the synthesis. Clearly indicate the labelled carbons in the resulting palmitate.
Calculate the total net gain in ATP and H2O molecules from the complete oxidation of this TAG. Assume that complete oxidation of the glycerol backbone yields +16ATP and +3H2O molecules.
Describe the activation of FA.
- Catalyzed by acyl-CoA synthetase (ACS)
- ACS links FA to CoA-SH in the cytosol - 2 step process.
- Step 1: AMP is attached to FA forming fatty acyl-adenylate
- Step 2: CoA-SH displaces AMP formin acyl-CoA
- This reaction uses 1 molecule of ATP but 2 ATP equivalents
- Both steps are reversible, but hydrolysis of pyrophosphate drives the overall reaction, so it becomes irreversible
Describe the first oxidation step of ß-oxidation.
Acyl-CoA dehydrogenase induces a double bond between C2 and C3 (α and ß carbons) using FAD as electron acceptor and generating FADH2 and trans-enoyl-CoA.
Describe the hydration step of ß-oxidation.
Enoyl-CoA hydratase adds water across the double bond resulting in L-ß-hydroxyacyl-CoA.
This reaction is stereospecific.
Describe the 2nd oxidation reaction in ß-oxidation.
ß-hydroxyacyl dehydrogenase converts L-ß-hydroxyacyl-CoA to ß-ketoacyl-CoA using NAD+ as an electron acceptor. (i.e., NAD+ is the oxidizing agent, and is itself reduced to NADH + H+).
Describe the last step in each cycle of ß-oxidation.
Thiolysis is catalyzed by thiolase which uses CoA-SH to cleave off acetyl-CoA, leaving acyl-CoA two carbons shorter.
The cycle continues until the acyl chain is completely broken down.
Note: The acyl group is attached to the new Co-A molecule.
How does 1 acetyl-CoA in the TCA cycle produce 10 ATP equivalents?
What is the overall reaction for complete oxidation of palmitoyl-CoA?
Palmitoyl-CoA (C16:0) + 7FAD + 7NAD+ + 7H2O + 7CoA-SH
>
8 acetyl-CoA + 7FADH2 + 7NADH + H+
Describe the ß-oxidation of unsaturated FAs. [2]
- Mono-unsaturated FAs require Δ3, Δ2 - enoyl-CoA isomerase to reposition the double bond to allow hydration by enoyl-CoA hydratase.
- FADH2 is not produced because C2-C3 bond is already oxidized, so there will be less ATP produced.
Compare FA synthesis and FA degradation. [8]
FA synthesis
- Occurs during times of plenty, (ATP high)
- Occurs in adipose, liver, mammary glands
- Occurs in the cytosol
- 2 carbons are added at a time
- Substrates: acetyl-CoA and malonyl-CoA
- Performed by one super enzyme complex with 7 domains and involves ACC as well.
- NADPH and ACP are used
- Intermediate: D-ß-hydroxyacyl-ACP
FA degradation
- Occurs during times of starvation (ATP low)
- Occurs in most tissues except the brain.
- Occurs in the mitochondrial matrix
- 2 carbons are removed at a time
- Products: acetyl-CoA (as well as propionyl-CoA for odd-numbered FAs)
- Requires multiple enzymes and involves ACS as well.
- FAD, NAD+, and CoA-SH are used.
- Intermediate: L-ß-hydroxyacyl-CoA
What are the 4 main targets for FA metabolism regulation?
- Acetyl-CoA carboxylase (ACC) - catalyzes the commited step of FA synthesis - regulated both allosterically and by hormone induced reversible phosphorylation.
- Carnitine Acyl Transferase I (CATI) - allosterically inhibited by malonyl-CoA (substrate of FA synthesis)
- ß-hydroxyacyl-CoA dehydrogenase - inhibited by NADH (product inhibition)
- Thiolase - inhibited by acetyl-CoA (product inhibition)
Describe the extensive role of acetyl-CoA carboxylase (the enzyme that catalyzes the committed step of FA synthesis) in the regulation of FA metabolism. [3]
-
ACC is regulated allosterically
- Citrate activates ACC: indicates that there is high enough activity of TCA cycle (high acetyl-CoA, high ATP)
- Palmitoyl-CoA inhibits ACC: indicates there is enough FA inside the cell
-
ACC is regulated by hormones via reversible phosphorylation
- Glucagon (released by pancreas α-cells when blood glucose is low) will activate via signalling cascade protein kinase A (PKA), which in turn phosphorylates (deactivates) ACC
- Insulin (released by pancreas ß-cells when blood glucose is high) will activate via signalling cascade phosphatase, which in turn dephosphorylates (activates) ACC
-
ATP/AMP levels can change phosphorylation status of ACC
- AMPK (AMP-dependent kinase) is activated by AMP (indicates low energy levels) or deactivated by ATP (indicates high energy levels).
- When AMPK is active it will phosphorylate ACC and deactivate FA synthesis.
Describe the role of CATI in regulation of FA metabolism.
Malonyl-CoA allosterically inhibits carnitine-acyl transferase I (CATI), so when FA synthesis is activated FA transport into mitochondria will be inhibited.
Describe the role of thiolase in regulation of FA metabolism.
Thiolase is inhibited by acetyl-CoA (product inhibition).
Describe the composition of cholesterol.
27 carbon molecule - all carbons are derived from acetyl-CoA
Describe the importance of cholesterol. [3]
- Stabilizes membrane fluidity
- Precursor for steroid hormones (e.g., testosterone, estrogen)
- Precursor of bile salts (e.g., taurocholic acid)
NOTE: excess dietary cholesterol can be deposited on the blood vessels and lead to atherosclerosis
Cholesterol sources. [2]
- Diet (mostly from animal products)
- Synthesis
Note: Mammals cannot break down cholesterol back to acetyl-CoA
Isoprene
Describe the first two reactions in the first major step of cholesterol synthesis.
IPPP/DMAPP synthesis - occurs in the cytosol
- Condensation of 2 acetyl-CoA by thiolase (reverse of thiolysis step of ß-oxidation) to form acetoacetyl-CoA (4C)
- Condensation of acetoacetyl-CoA and acetyl-CoA by HMG-CoA synthase to form HMG-CoA (6C)
ß-hydroxy-ß-methylglutaryl-CoA
(HMG-CoA)
ß-hydroxy-ß-methylglutaryl-CoA
(HMG-CoA)
Acetoacetate (ketone body)
Cholesterol
Acetoacetyl-CoA
2 acetyl-CoA
ß-hydroxy-ß-methylglutaryl-CoA
(HMG-CoA)
Describe the third reaction in the first major step of cholesterol synthesis.
Reduction of HMG-CoA by HMG-CoA reductase using NADPH to form mevalonate (6C).
This is the irreversible (committed step) and therefore a site of regulation.
This step and all further steps of de novo cholesterol synthesis occur on the outer leaflet of the smooth endoplasmic reticulum.
Describe what follows the third reaction of the first major step of cholesterol synthesis.
Through a series of reactions mevalonate is converted to activated isoprenoids: isopentenyl pyrophosphate (IPPP) or dimethylallyl pyrophosphate (DMAPP).
3 ATPs are consumed and 1 carbon is lost in the form of CO2.
Name two activated isoprenes.
IPPP and DMAPP
Mevalonate
5-Phosphomevalonate
5-Pyrophosphomevalonate
What is the reaction intermediate directly preceding IPPP/DMAPP production in cholesterol synthesis?
3-phospho-5-pyrophosphomevalonate
Δ3-Isopentenyl pyrophosphate (IPPP)
Dimethylallyl pyrophosphate (DMAPP)
Geranyl pyrophosphate
Farnesyl pyrophosphate
Describe the 2nd major step of cholesterol synthesis. [3]
Synthesis of squalene
- Condensation of IPPP and DMAPP in a head-to-tail arrangement by dimethylallyl pyrophosphate prenyl transferase to form geranyl pyrophosphate (10C)
- Condensation of geranyl pyrophosphate and IPPP in a head-to-tail arrangement by prenyl transferase to form farnesyl pyrophosphate (15C)
- Condensation of two farnesyl pyrophosphates in head-to-head orientation by squalene synthase using NADPH to form squalene (30C)
All three condensation reactions that occur during the synthesis of squalene occur in a head-to-tail arrangement.
True or false?
False.
- Dimethylallyl pyrophosphate prenyl transferase reaction is IPPP and DMAPP in head-to-tail arrangement.
- Prenyl transferase reaction is geranyl pyrophosphate and Δ3-Isopentenyl pyrophosphate (IPPP) in head-to-tail arrangement.
- Squalene synthase reaction is two farnesyl pyrophosphates (15C+15C) in head-to head arrangement.
Squalene 2,3-epoxide
Squalene 2,3-epoxide
Lanosterol
Describe the 3rd major step of de novo cholesterol synthesis. [2]
- Cyclization of squalene via a series of reactions to form lanosterol.
- Hydroxyl group is added which requires NADPH and O2.
- Lanosterol is converted to cholesterol (19 reactions!) with the removal of 3 methyl groups (30C>27C) and using NADPH as a reducing agent.
Describe local regulation of HMG-CoA. [3]
-
Transcription of HMG-CoA reductase is controlled by cholesterol
- Sterol regulatory element (SRE) is a sequence of DNA near the HMG-CoA reductase gene that can be recognized by SRE binding protein (SREBP)
- When cholesterol levels are high SREBP is anchored in the ER membrane
- When cholesterol levels are low SREBP is translocated to the Golgi, where it is cleaved twice by proteases and freed from the membrane.
- Free SREBP diffuses to the nucleus and activates transcription of HMG-CoA reductase.
- Note: Free SREBP is rapidly degraded to prevent overproduction of cholesterol
- Translation of HMG-CoA reductase mRNA is inhibited by mevalonate derivatives
- Degradation of HMG-CoA reductase is stimulated by lanosterol and 25-hydroxycholesterol
25-hydroxycholesterol
25-hydroxycholesterol
25-hydroxycholesterol
25-hydroxycholesterol
What is SREBP?
- Sterol regulatory element (SRE) is a sequence of DNA near the HMG-CoA reductase gene that can be recognized by SRE binding protein (SREBP).
- When cholesterol levels are high SREBP is anchored in the ER membrane.
- When cholesterol levels are low SREBP is translocated to the Golgi, where it is cleaved twice by proteases and freed from the membrane.
- Free SREBP diffuses to the nucleus and activates transcription of HMG-CoA reductase.
- Free SREBP is rapidly degraded to prevent overproduction of cholesterol.
Describe the synthesis of cholesteryl esters.
- FAs can be attached to the OH group of cholesterol making it more hydrophobic.
- Cholesteryl esters are used to store and transport cholesterol (lipoproteins).
- Synthesized in the liver by acyl-CoA cholesterol transferase (ACAT) using FA-CoA and producing CoA-SH
- Also synthesized are high density lipoprotein particles (HDL) by lecithin cholesterol acyl transferase (LCAT) using phosphatidylcholine and producing lysophosphatidylcholine.
Cholesteryl oleate
(A cholesteryl ester)
Cholesteryl Ester (with C17:1 acyl group)
Lysophosphatidylcholine
Describe bile salts and their importance. [3]
- Polar derivatives of cholesterol synthesized in the liver and stored in the gallbladder.
- Help to emulsify dietary lipids in small intestines
- Major way to excrete cholesterol
What is the precursor of all steroid hormones?
Pregnenolone (C21)
Pregnenolone (C21) - precursor to all steroid hormones
Name this molecule and describe its importance. [2]
Progesterone
- Involved in ovarian cycle
- Prepares lining of uterus for ovum implantation
Name this molecule and describe its importance.
Aldosterone
- Causes kidney to reabsorb Na+ and excrete K+
- Increases blood volume and pressure
Name this molecule and describe its importance.
Testosterone
- Causes development of male
- And female secondary sexual characteristics
Name this molecule and describe its importance.
Estradiol
- Causes development of male
- And female secondary sexual characteristics
Cortisol
- Inhibits inflammation
- Promotes gluconeogenesis and glycogen formation
- Promotes fat and protein breakdown
Name this molecule and describe its importance.
Cortisol
- Inhibits inflammation
- Promotes gluconeogenesis and glycogen formation
- Promotes fat and protein breakdown
Name this molecule and describe its importance.
Progesterone
- Involved in ovarian cycle
- Prepares lining of uterus for ovum implantation
Name this molecule and describe its importance.
Aldosterone
- Causes kidney to reabsorb Na+ and excrete K+
- Increases blood volume and pressure
Name this molecule and describe its importance.
Testosterone
- Causes development of male
- And female secondary sexual characteristics
Name this molecule and describe its importance.
Estradiol
- Causes development of male
- And female secondary sexual characteristics
Name the 5 major classes of steroid hormones and give examples of each.
- Progestagens (e.g., progesterone)
- Mineralocorticoids (e.g., aldosterone)
- Glucocorticoids (e.g., cortisol)
- Androgens (e.g., Testosterone)
- Estrogens (e.g., Estradiol)
Where are bile salts synthesized, stored and what is their major purpose?
- They are polar derivatives of cholesterol synthesized in the liver and stored in the gallbladder.
- They help to emulsify lipids in small intestines.
Describe the digestion of dietary lipids.
Lipids (TAG) are first emulsified by bile (bile salts + choesteryl esters) into micelles and then broken down by lipases to monoacylglycerol (MAG) and 2 FAs.
Describe the activity of intestinal lipase and lipoprotein lipase.
Water is used to break down TAG to MAG.
Describe the absorption of lipids. [3]
- MAG and FAs are absorbed via facilitated diffusion using membrane transporters.
- Inside intestinal cells TAG are reassembled and packaged with cholesterol and apolipoproteins into lipoprotein particles called chylomicrons.
- Chylomicrons are released via lacteal (lymphatic system capillary) and travel via lymphatic system bypassing liver.
Describe the distribution and uptake by tissues of lipids. [2]
- Lymphatic system carrying chylomicrons merges with bloodstream.
- Chylomicrons are distributed by bloodstream to tissues and where endothelial cells express tissue lipoprotein lipase, cleaves TAG to 3 FAs and glycerol, which in turn are picked up by tissues and used for energy (e.g., muscle) or stored (re-esterified to TAG in adipose tissue).
Name 4 types of lipoprotein particles.
- Chylomicrons: large and very low density
- Very low density lipoprotein (VLDL)
- Low density lipoprotein (LDL)
- High density lipoprotein (HDL)
What are chylomicrons?
Large and very low density lipoprotein particles that contain primarily dietary TAG and small amounts of cholesterol. Remnants of chylomicrons are picked up by the liver.
What is LDL?
- Low density lipoprotein particle that transports endogenous cholesterol from liver to tissues.
- ApoB100 in LDL is recognized by LDL receptor in tissues and picked up by endocytosis.
- ‘Bad cholesterol’ - high levels are associated with obesity, diabetes, and heart disease.
- Excessive amounts can be deposited on capillary walls leading to atherosclerosis.
- Image on other side: peripheral tissue uptake of cholesterol by LDL (ApoB100)-receptor mediated endocytosis
What is VLDL?
- Very low density lipoprotein - transport of endogenously synthesized TAG from the liver to peripheral tissues including adipocytes.
- Remnants of VLDL are converted to LDL or picked up by the liver in the form of IDL.
- e.g., excessive (glucose>acetyl-CoA>FAs)liver >VLDL>adipose tissue
Describe digestion of dietary proteins. [3]
- An interplay between organs and hormones results in activation of dietary proteases (e.g., chymotrypsin) that hydrolyze proteins to individual amino acids
- AA are absorbed into the bloodstream, go via portal vein (vein that connects intestines and liver) and then distributed among tissues.
- Picked up by AA transporters
What is the purpose of endogenous protein breakdown. [4]
- To breakdown proteins that are not needed anymore - recycling.
- To remove damaged or misfolded proteins
- Inactivation of enzymes in metabolic pathways - regulatory (e.g., HMG-CoA reductase, cyclins)
- For energy - only during prolonged starvation
All proteins are continually synthesized and degraded - protein turnover.
What is ubiquitin?
- Proteins tagged with a small (8.5kDa) protein called ubiquitin are targeted to degradation.
- C-terminus of ubiquitin (Ub) forms isopeptide bond with epsilon-amino group of Lysine’s side chain in target protein
How are proteins tagged for destruction?
- Ubiquitination of proteins.
- C-terminus of ubiquitin forms isopeptide bond with epsilon-amino group of Lysine’s side chain in target protein.
Describe the formation of an isopeptide bond in protein ubiquitination.
Multistep process requiring 3 enzymes
- E1 - Ub-activating enzyme (UbA) 1 gene
- E2 - Ub-conjugating enzyme (UbC) several genes
- C3 - Ub-ligase (UbL) - several hundred genes
Step 1a - E1 adenylates Ub forming Ub-AMP (2 ATP equivalents are used)
Step 1b - Ub is transferred onto Cys residue of E1.
Step 2 - E2 transfers Ub from E1 to E2.
Step 3a - E3 recognizes and binds target protein and Ub-E2
Step 3b - E3 transfers Ub from E2 to Lys of target protein forming isopeptide bond.
Note: sometimes other AA can be ubiquitinated (e.g., Cys)
How many ubiquitin need to be attached to a target protein for degradation?
4 on average (8 ATP cost)
Note: Ubiquitin is a protein with a Lys residue that can be tagged by another Ubiquitin.
What is the N-terminal rule?
If a protein has Met at the N-end then it usually has a longer half-life, and if it has another AA (e.g., Arg), it has a shorter half-life.
Describe protein proteolysis in proteasome. [5]
- 26S complex (64 subunits) that degrades Ub-proteins
- Forms a hollow tube open at both ends.
- Two 19S caps recognize Ub-proteins and denature it
- Denatured proteins are fed into the ‘tube’ (requires ATP), while Ub is cleaved off for reuse.
- The ‘tube’ (20S core) degrades target proteins into small peptides that are released into the cytosol where they can be further degraded by cellular peptidases
How do aminotransferases work? [3]
- All aminotransferases have the same prosthetic group - PLP (pyridoxal phosphate) - tightly bound in the active site by non-covalent interactions.
- PLP is derived from pyridoxine (B6) and has a highly reactive aldehyde group
- Aldehyde group of PLP will react with amino group of AAs forming aldimine (Schiff base)
Describe the resting state of PLP.
In resting state the aldehyde group of PLP is in Schiff base with amino group of enzyme’s Lysine: Lys(ε)-PLP
What is the mechanism of Schiff base formation?
Describe transport of amino groups to the liver. [2]
-
Glutamine: peripheral cells that produce glutamate via transamination and NH4+ from metabolic reactions.
- Glu + NH4+ > Gln + H2O (uses ATP)
- Above reaction done by glutamine synthetase
-
Alanine: (Cahill cycle -glucose-alanine cycle)
- In muscle during heavy exercise exessive amounts of pyruvate are produced along with excessive amounts of NH3 produced from AA catabolism.
- Pyruvate can be transaminated to form alanine and then delivered to the liver where it forms back the pyruvate then used for gluconeogenesis and glutamate (transamination again)
What happens to Glu in the liver?
Glu > alpha-ketoglutarate + NH4+
Oxidative deamination catalyzed by glutamate dehydrogenase using NAD+ or NADP in the mitochondrial matrix.
What happens to Gln in the liver?
Gln H2O > Glu + NH4+
Deamination catalyzed by glutaminase in the mitochondrial matrix
What happens to free ammonia in the liver?
- Excreted directly - only in aquatic animals
- Form urea - land animals such as mammals or amphibians
- Form uric acid - land animals such as reptiles and birds
Describe the preparatory step of the urea cycle.
- Synthesis of carbamoyl phosphate from ammonia and bicarbonate
- Catalyzed by carbamoyl phosphate synthetase (CPS-1)
- Requires 2 ATP
- Occurs in the mitochondrial matrix - 3 steps:
- Bicarbonate is phosphorylated by ATP to form carboxyphosphate (highly reactive)
- NH3 displaces the phosphate to form carbamate
- Carbamate is phosphorylated by ATP to form carbamoyl phosphate
Carboxyphosphate
Carboxyphosphate
Describe the urea cycle. [4]
-
Carbamoyl phosphate reacts with amino acid ornithine to form amino acid citrulline in the mitochondrial matrix.
- Catalyzed by ornithine transcarbamylase (OTC)
- Pi is displaced
- Citrulline is transported to the cytosol
-
Citrulline is activated by ATP and then reacts with aspartate to form argininosuccinate
- Catalyzed by argininosuccinate synthase
- AMP and PP are formed (2 ATP equivalents are used)
-
Argininosuccinate is cleaved to form fumarate and arginine
- Catalyzed by argininosuccinase
-
Arginine is hydrolyzed to generate urea and ornithine
- Catalyzed by arginase
- Ornithine is transported back to the mitochondrial matrix
Fumarate
Ornithine
Citrulline
Aspartate
Argininosuccinate
Fumarate
What happens to the fumarate produced by the urea cycle?
It can be converted to malate first by cytoplasmic fumarase, and then malate can be transported back to the mitochondrial matrix where it is converted to oxaloacetate by malate dehydrogenase.
What is argininosuccinase deficiency and how may it be treated?
Argininosuccinate will accumulate if argininosuccinase is deficient. Since it cannot be cleaved to Arginine and fumarate the urea cycle stalls and ammonia will start to build up.
Treatment:
- Arginine and aspartate supplementation - bypass block and argininosuccinate is excreted in the urine. Allows ammonia and Aspartate to flow again.
- Eat very low protein diet.
Describe short term regulation of the urea cycle. [4]
- Formation of N-acetylglutamate from acetyl-CoA and glutamate by N-acetylglutamate synthase
- N-acetylglutamate is an allosteric activator of CPS-1
- N-acetylglutamate is formed when Glutamate levels are high
- Arginine is a positive regulator of N-acetylglutamate synthase
All carbon skeletons (alpha-ketoacids) are broken down directly or in several steps into one of 7 intermediates. These intermediates are:
- Pyruvate
- alpha-ketoglutarate
- Succinyl-CoA
- Fumarate
- Oxaloacetate
- Acetyl-CoA
- Acetoacetyl-CoA
All 7 can feed into the TCA cycle.
1 -5: glucogenic AA, meaning we can use them to make glucose via gluconeogenesis
6 and 7: cannot be converted to glucose, but can be converted to ketone bodies
Describe the catabolism of Phenylalanine and Tyrosine.
- Both are gluconeogenic and ketogenic
- Transamination happens on the second step
Overview
- Phe is converted to Tyr by phenylalanine hydroxlyase (PAH) using O2 and cofactors tetrahydrobiopterin (THB) and NADH. As a result Phe is hydroxylated and THB is oxidized to dihydrobiopterin (DHB) and require another enzyme DHB reductase to convert it back to THB.
- Transamination of Tyr by tyrosine aminotransferase (TAT) using alpha-ketoglutarate as acceptor of amino group.
- Reactions 3-7 the carbon skeleton (hydroxyphenylpyruvate) is decarboxylated, hydroxylated, opened, rearranged, and finally split to fumarate and acetoacetyl-CoA.
Describe the reaction catalyzed by phenylalanine hydroxylase.
- Phe is converted to Try using O2 and cofactors THB and NADH.
- As a result Phe is hydroxylated and THB is oxidized to dihydrobiopterin (DHB).
- DHB reductase is another enzyme that converts DHB back to THB
How is DHB converted to THB?
DHB reductase and cofactor NADH.
How is phenylalanine converted to p-Hydroxyphenylpyruvate?
What happens to the hydroxyphenylpyruvate?
Overview: catabolism of phenylalanine
- Phe is converted to Tyr by phenylalanine hydroxylase (PAH) using O2 and cofactors tetrahydrobiopterin (THB) and NADH. As a result Phe is hydroxylated and THB is oxidized to dihydrobiopterin (DHB) and require another enzyme DHB reductase to convert it back to THB.
- Transamination of Tyr by tyrosine aminotransferase (TAT) using α-ketoglutarate as acceptor of amino group
- In 5 rxns (3-7) carbon skeleton (hydroxyphenylpyruvate) is decarboxylated, hydroxylated, opened, rearranged, and finally split to fumarate and acetoacetyl-CoA
Describe potential defects in Phe and Tyr catabolism.
- Mutation in phenylalanine hydroxylase (phenylketonurio -PKU) leads to build-up of Phe, that will undergo oxidative deamination to produce phenylpyruvate.
- Phe and phenylpyruvate are toxic in high concentration
- Symptoms: mental retardation and death (if untreated)
- Treatment: diet with no Phe and Tyr supplementation
- Note: Tyrosine is precursor to neurotransmitters (dopamine, norepinephrine, epinephrine) and for pigment melanin.
Draw the reaction catalyzed by alanine aminotransferase (AAT).
Draw the biosynthesis of Asp and Asn.
Name 3 ketone bodies.
Acetone
Acetoacetate
D-ß-hydroxybutyrate
Describe nutrients during fasting and starvation. [4]
- When glucose is depleted we start using glycogen, and then gluconeogenesis (from glucogenic AAs) is activated.
- Proteins are not normally used for energy, but during prolonged starvation when gluconeogenesis is activated we see protein breakdown and glucogenic AAs are transaminated and carbon skeletons are used as substrate for gluconeogenesis.
- During starvation TAG are broken down into glycerol and FAs, and then FAs are carried by albumin to the tissues (liver, muscle, heart) to be used for energy.
- Ketone bodies are synthesized by liver from acetyl-CoA, and then released to the bloodstream where they are picked up by the brain and heart, where they are converted back to acetyl-CoA and used for energy via TCA cycle. Too many ketones (e.g., diabetes) is toxic.
Ketone bodies are toxic.
True or false?
True - in large amounts (e.g., diabetes)
What are the four phases of fasting?
- C16:0 or C16:0 or 16:0
- C18:1 cis Δ9 or C18:1 cis Δ9 or 18:1 cis Δ9
- C18:3 cis Δ9,12,15 or C18:3 cis Δ9,12,15 or 18:3 cis Δ9,12,15
Phytol, a long chain alcohol, appears as an ester in plants. When consumed as part of the diet, phytol is converted to phytanic acid. Refsum’s disease is a neurological disorder related to the fact that phytanic acid accumulated in the membranes of nerve cells and causes disruption of the cell membrane.
Based on your knowledge of lipid membrane structure, what general effects of phytanic acid on these membranes would be observed?
When phytanic acid (branched FA) accumulates in nerve cells there is a disruption of van de waals forces which leads to looser packing of the fatty acids in the cell membrane.
This leads to a decrease in melting temperature and hence easier disruption of the nerve cell membranes.
Which of the following statements are true about this lipid?
A. This lipid molecule does not contain a “sphingosine” backbone. B. This lipid molecule contains a defined glycerol backbone.
C. The fatty acid moiety is attached via an amide linkage.
D. If “A” were substituted with a hydrogen, this molecule would be labeled as “Sphingomyelin.”
E. If “A” were substituted with phosphocholine, this molecule would be labeled as a “cerebroside.”
C. The fatty acid moiety is attached via an amide linkage.
Below is the melting curve for a membrane composed entirely of glycerophospholipids (containing only saturated fatty acyl chains). Draw the melting curve for the same membrane with the addition of lipids mentioned in part A, B and C. Also explain the mechanism of action for A, B, and C.
A) Some glycerophospholipids with unsaturated fatty acyl chains
Melting temperature is lowered – curve shifts to the left. Introducing glycerophospholipids with unsaturated fatty acyl chains would lead to looser packing of the lipids in a membrane, which in turn would decrease melting temperature.
Below is the melting curve for a membrane composed entirely of glycerophospholipids (containing only saturated fatty acyl chains). Draw the melting curve for the same membrane with the addition of lipids mentioned in part B. Also explain the mechanism of action for B.
B) Some glycerophospholipids with slightly longer fatty acyl chains
Melting temperature is increased – curve shifts to the right. Introducing glycerophospholipids with longer fatty acyl chains would lead to tighter packing of the lipids in a membrane, which in turn would increase melting temperature.
Below is the melting curve for a membrane composed entirely of glycerophospholipids (containing only saturated fatty acyl chains). Draw the melting curve for the same membrane with the addition of lipids mentioned in part C. Also explain the mechanism of action for C.
C. Cholesterol.
Cholesterol acts like a fluidity buffer: Below Tm – cholesterol disrupts close packing of acyl chains and increases membrane fluidity. Above Tm – cholesterol constrains motion of acyl chains and decreases membrane fluidity. It essentially broadens the phase transition.
The carbonyl carbon of acetyl-CoA is radioactively labelled with 14C. Suppose acetyl-CoA carboxylase (ACC) uses this radioactive acetyl-CoA to synthesize malonyl-CoA.
Draw the resulting malonyl-CoA and clearly label the location of the radioactive carbon.
The carbonyl carbon of acetyl-CoA is radioactively labelled with 14C. Suppose fatty acid synthase uses these radioactive substrates for the first round of fatty acid synthesis.
Draw the resulting 4-carbon intermediate that is bound onto the acyl carrier protein (ACP) and clearly label the location of the radioactive carbon.
The carbonyl carbon of acetyl-CoA is radioactively labelled with 14C. Suppose ACC has been mutated in such a way that it is no longer capable of functioning. Excess unlabeled malonyl-CoA is now added into the cytosol for fatty acid synthesis.
Draw the final synthesized fatty acid product and clearly label the location of the radioactive carbon.
Palmitate (C16:0) is in the process of being synthesized on the enzyme fatty acid synthase. A “snap shot” of the enzyme has been taken and a chemical structure is distinguishable on the acyl carrier protein (ACP) of FA synthase (observe Step B).
A. Give the name of the structure (using Cn:X format) present at Step B of the FA synthesis process.
B. With your knowledge of FA synthesis, please draw the chemical structure X in Step A (the preceding step) of the FA synthesis process. Note: You do not need to include or know the name of this chemical structure.
A. C8:1 trans Δ2-ACP
B. See image
With your knowledge of FA synthesis, please draw the chemical structure “Y” in Step C of the FA synthesis process. Please include the name of this chemical structure (using Cn:X format).
How many of the following reactions occured between A and B?
Oxidations
Hydrations
Thiolysis
Reductions
Dehydrations
Isomerization
Oxidations - 4
Hydrations - 2
Thiolysis - 3
Reductions - 0
Dehydrations - 0
Isomerization - 1
How many ATP and water are generated going from A to B?
38 ATP
8 H2O
The three most common fatty acyl groups stored in human adipose are derived from palmitate, oleate and linoleate. Below in the structure of a TAG containing one of each acyl chain. The molecular weight of this specific TAG is 861 g/mol with a formula of C55H104O6.
A 140kg man loses 60kg of fat! After his diet he asks you” “where did all my mass go”’ Give the biochemical explanation to his question. For full marks attempt to quantitate you answer. Note: glycerol can indirectly enter glycolysis and in turn be converted to pyruvate.
When a C20:2 cis Δ9,15 - CoA undergoes complete oxidation, what is the net gain in ATP and water molecules.
How many C21:1 cis Δ9 fatty acids are required to supply 12,000 ATP?
Calculate the number of water molecules generated from the oxidation of the fatty acids required above.
Suppose an individual is undergoing severe starvation. This individual’s liver cell contains 32 molecules of C18:1 cis Δ9 and 26 molecules of C16:0.
What is the maximum number of ATP molecules and water molecules that could potentially be generated in the liver cell if all of the FA are oxidized to CO2 and water.
Pelargonate is a 9-carbon saturated fatty acid found in plants and many fruits that we commonly eat.
What is the net ATP and water molecule gain or cost when it is completely oxidized to CO2 and H2O?
Waxes are important energy storage molecules for zooplankton – microscopic animals found in our oceans that feed on photosynthetic phytoplankton. Waxes also provide a huge worldwide energy source for many marine animals making plankton one of the pillars of the marine food chain. The 18m long 100-ton right whale feeds almost exclusively on plankton! What are the ATP and H2O yields for the complete oxidation of the following 36-carbon wax to CO2 and water for a right whale?
- Note 1: An extracellular lipase can hydrolyze the ester linkage. The cell absorbs the resulting two products.*
- Note 2: Fatty alcohols are oxidized to corresponding fatty acids by certain cytoplasmic enzymes.*
Suppose that glutamate labeled with 15N undergoes degradation in the liver of a rat. The amino group then enters the urea cycle to produce a molecule of urea. Which atoms of the following metabolites will the isotopes be found? Assume in this case that aspartate must be synthesized from glutamate and that unlabelled ornithine is already abundant. Mark 15N atoms with an asterisk (*).
Ornithine
Citrulline
Aspartate
Urea
Citrulline
Ornithine
Ornithine
A child is admitted to the hospital due to progressive lethargy and nausea. Which of the following defects could account for the results of the blood test?
A. arginase deficiency
B. argininosuccinase deficiency
C. ornithine transporter deficiency
D. carbamoyl phosphate synthetase-I deficiency
E. argininosuccinate synthetase deficiency
E. argininosuccinate synthetase deficiency
A two-year-old child was taken to the hospital. His mother said he vomited frequently, especially after feedings. The child’s weight and physical development were below normal. His hair, although dark, contained patches of white. A urine sample treated with ferric chloride (FeCl3) gave a green colour characteristic of the presence of phenylpyruvate. Quantitative analysis of urine samples gave:
A. What are the three compounds?
B. What type of enzyme is converting compound 2 into compound 3?
C. Suggest which enzyme might be deficient in this child and propose a treatment.
A. Phenylalanine, pyruvate, alanine
B. Aminotransferase
C. Accumulation of Phe is a symptom of PKU. Treatment is to limit Phe intake and supplement tyrosine to allow the pathway to proceed.
A pharmaceutical company discussed the possibility of using a HMG-CoA synthase inhibitor to reduce blood cholesterol levels. Biochemists in the company raised concerns that the drug could be quite dangerous under a certain metabolic condition, such as people using fat as a major energy source due to a low carbohydrate and high fat diet.
Discuss why the drug could be dangerous.
The drug would also inhibit ketone body synthesis in the liver because the same step is part of ketone body synthesis. When fat is used as a major energy source, organs like the brain, heart, skeletal muscles and renal cortex rely on the supply of ketone bodies from the liver for its function.
