BIOC 302 1

In the presence of glucagon, the white adipocyte glucagon receptor activates adenylyl cyclase that converts ATP to cAMP. Increases in the cAMP concentration activate protein kinase A (PKA) that, in turn, phosphorylates its downstream targets to orchestrate lipid breakdown. List three key enzymes that mediate the breakdown of triacylglycerol (TAG) into fatty acids (FA), and briefly describe their functions.

ATGL (Adipose Triglyceride Lipase): converts TAG to DAG and FA. HSL (Hormone Sensitive Lipase): converts DAG to MAG and FA. MGL (Monoacyl glycerol lipase): converts MAG to glycerol and FA.

List how Protein Kinase A (PKA) is activated and deactivated by other kinases or small signaling molecules. Also name the protein that it phosphorylates in glycogen metabolism.

Activated by cAMP binding to regulatory subunit Deactivated by loss of cAMP due to phosphodiesterase activity causing rebinding of the catalytic subunit to the regulatory subunit inhibiting its activity. Phosphorylates phosphorylase b kinase to activate it. It also phosphorylates GM and inhibitory proteins on the glycogen particle that cause PP1 to leave the particle

List how Protein Kinase B (PKB) is activated and deactivated by other kinases or small signaling molecules. Also name the protein that it phosphorylates in glycogen metabolism.

Activated by interactions with PDK-1 which is activated by the binding of PIP3 - any general answer going all the way back to the insulin receptor and IRS-1, PI-3K etc. Deactivated by loss of insulin signal, IRS-1 phosphorylation, etc. to loss of PDK-1 activity - Again accept almost any reasonable answer since it is not entirely clear how this works. It phosphorylates Glycogen Synthase Kinase 3 (GSK3)

List how Phosphorylase b kinase is activated and deactivated by other kinases or small signaling molecules. Also name the protein that it phosphorylates in glycogen metabolism.

Activated by phosphorylation by PKA Deactivated by protein phosphatase 1 (PP1) on the glycogen particle. Phosphorylates glycogen phosphorylase to activate it for glycogen breakdown.

Describe the steps involved in fat (triglyceride) mobilization and transport from adipocytes to liver.

Activation of lipases by protein kinase A converts triglycerides to fatty acids and glycerol in the adipocyte. Fatty acids bind to albumin for transport in the serum. Glycerol diffuses from adipocyte and is converted to glycerol-3-phosphate in liver for use in glycolysis and TCA cycle.

Once inside the mitochondria all three fatty acids are completely oxidized to 6 CO2 molecules, initially by the β oxidation pathway and then completely by the TCA cycle. Indicate the location of the four enzymes that lead to β-oxidation and production of acetylCoA (i.e. matrix or inner membrane), write out each reaction with cofactors using simple structures or words, and name each enzyme involved in the process.

All the enzymes for β-oxidation of moderately long fatty acylCoAs are located in the matrix of the mitochondrion.

Asparagine is a glucogenic amino acid. Describe the pathways and important enzymes that yield glucose from asparagine. How many molecules of glucose does one asparagine yield (i.e., what is the stoichiometry)?

Asparagine → Oxaloacetate (2) Oxaloacetate → (1) Glucose (Gluconeogenesis) 1 Asparagine per Oxaloacetate and 2 Oxaloacetates are required per glucose, so a single Asparagine molecule will yield 0.5 glucose Additional points could be given for knowing Asparagine → Aspartate → Oxaloacetate (via asparaginase and aspartate aminotransferase)

Explain how caffeine prolongs activation of glycogen breakdown in this GPCR pathway.

Caffeine inhibits the phosphodiesterase that converts cAMP to simple AMP. As result, cAMP levels and PKA activity stay high even, after the adrenaline signal in the blood goes away and adenylate cyclase activity stops.

How are fatty acids transported into the mitochondrion? How is simultaneous use of cytoplasmic fatty acyl-CoA in both fatty acid synthesis and β-oxidation prevented?

Cytoplasmic fatty acids are first converted to fatty acyl-coA. In the outer mitochondrial membrane the fatty acid group is transferred from fatty acyl-CoA to carnitine by carnitine acyl transferase I. Fatty acyl-carnitine reaches the inner mitochondrial membrane and the fatty acyl group is again transferred to form fatty acyl-CoA in the mitochondrial matrix by carnitine acyl transferase II. Malonyl- CoA, a key intermediate in fatty acid synthesis, strongly inhibits carnitine acyl transferase I preventing fatty acyl-CoA use in β-oxidation while fatty acid synthesis is occurring in the cytoplasm.

Name three types of amino acid transformations that can be catalyzed by pyridoxal phosphate (PLP) containing enzymes. What feature of pyridoxal phosphate's structure makes it so versatile in supporting these different types of reactions?

Deamination/transamination, decarboxylation, racemization, β-elimination, γ-elimination and C-C bond cleavage or addition are all acceptable answers. The conjugated bond system or resonance of the pyridine ring is the key feature of PLP that allows it to support these diverse reactions.

Describe in words how this oil is digested in the small intestine, how the fatty acids are packaged for transport into the blood stream, and how they are taken up by adipose tissue cells for storage. Name at least two lipases that are involved in these processes and any necessary transport proteins.

Fats and oils pass through the stomach and become emulsified in the small intestine by bile salts and then are broken down by pancreatic lipases. The free fatty acids are taken up by intestinal villi cells where they are re- esterified into triacylglycerols. These new oils are then packaged into large chylomicron lipoprotein particles which enter the lymphatic system before entering the blood stream. The triacylglycerols in the chylomicrons are broken down in blood vessels surrounding adipocytes by clearing factor or lipoprotein lipase that recognizes bound ApoC. The released fatty acids are repackaged a second time into lipid droplets in adipose tissue.

Once phosphoenolpyruvate is formed in the cytoplasm it can be converted through a series of readily reversible steps to fructose-1,6-bisphosphate. How is fructose-1,6-bisphosphate converted to glucose? How is the glycolytic breakdown of these intermediates avoided?

Fructose-1,6-bisphosphate is converted to fructose-6-phosphate by Fructose bisphosphatase-1 (FBP-1). Reconversion to F-1,6-BisP by Phosphofructokinase-1 is inhibited by elevated ATP levels and loss of allosteric activation by Fructose-2,6-bisphosphate. F-2,6-BisP formation by PFK-2/FBP- 2 is inhibited by glucagon-activated phosphorylation. Fructose-6-phosphate is reversibly converted glucose-6-phosphate. Glucose-6-phosphate is transported to the endoplasmic reticulum and converted to glucose by glucose-6- phosphatase. Movement into this compartment keeps cytoplasmic glucose-6- phosphate from being reconverted back to fructose-6-phosphate.

Why are fumarase and malate dehydrogenase activities required for the Urea Cycle to function smoothly? Sketch out the Urea Cycle and parts of the TCA cycle to show why these enzymes are needed for net urea synthesis.

Fumarase and malate dehydrogenase are part of the aspartate-arginiosuccinate shunt of the citric acid cycle that regenerates Asp for addition of its α-amino group to citrulline.

Perilipin is one of the downstream target proteins of PKA in white adipocytes. A partial out-of-frame mutation disrupted the phosphorylation site/domain of this protein, and the adipocytes started to accumulate an unusual amount of lipid droplets within the cell. Explain the molecular mechanics behind the phenotypic change, and identify the protein(s) directly involved with the function/action of perilipin.

If PKA cannot phosphorylate perilipin, CGI is not released from it. As CGI is held on to perilipin, ATGL cannot be activated to initiate TAG breakdown. As the result, fat droplets accumulate.

Write out the "extra" enzymatic reactions, which allow the conversion of acetylCoA to glucose in plants via succinate and which are missing in animals (include names and any cofactors if necessary)?

In animals, there is no conversion of acetylCoA into C4 acids because the intact TCA cycle always involves two decarboxylation reactions so the net result is combustion and not condensation. However, in plants and most bacteria, the decarboxylation steps are bypassed via the glyoxylate shunt or cycle. In this pathway, acetylCoA condenses with oxaloacetate(OAA) to form citrate, which then isomerizes to isocitrate. Instead of β-keto acid decarboxylation and dehydrogenation, isocitrate undergoes de- aldolization to form succinate and glyoxylate in a reaction catalyzed by isocitrate lyase. Then glyoxylate condenses with another acetylCoA to form malate by malate synthase. Malate dehydrogenase then regenerates OAA and the cycle keeps going, converting 2 acetylCoAs into succinate and 2CoASH.

Under conditions of very high muscle activity and amino acid oxidation, ammonia is released into urine in the kidneys when the urea cycle in both the liver and kidneys cannot keep up with ammonia production. What enzyme(s) generates the ammonia in the kidneys?

In the kidneys, glutamine is broken down to glutamate by glutaminase, which hydrolyzes the γ amide on the side chain to produce free ammonia and glutamate. Alanine in the blood will be transaminated, transferring the α-amino group to α-keto glutarate to produce glutamate. If the urea cycle is not fast enough, ammonia is released from glutamate dehydrogenase and will be excreted directly into urine in the kidneys.

At which steps of the Citric Acid Cycle are CO2's lost?

Isocitrate dehydrogenase or IDH; α-ketoglutarate dehydrogenase

Alanine and lactate have identical oxidation states. What is the net ATP yield for the complete oxidation (to CO2 and H2O) of lactate vs. alanine?

Lactate and alanine are both converted to pyruvate. Lactate Dehydrogenase generates 1 NADH yielding 2.5 ATP equivalents. Pyruvate oxidation through the TCA cycle yields 12.5 ATP equivalents (4 NADH (10 ATP) + 1 FADH2 (1.5 ATP) + 1 GTP). Therefore, the total for lactate is 15 ATP. Alanine is converted to pyruvate by alanine aminotransferase at no ATP or NADH cost/yield. However, in the case of alanine recall that in the urea cycle it costs 4 ATP to produce 1 urea molecule or 2 ATP/Ammonia. Therefore, the net yield for alanine is 10.5 ATP.

Name three amino acids that can produce acetoacetate. Acetoacetate, like oxaloacetate, is an oxidized 4 carbon substrate. Why can oxaloacetate be used to make glucose and why can't acetoacetate be used for this purpose?

Leucine, lysine, tryptophan, phenylalanine and tyrosine all can produce acetoacetate. OAA can be converted to phosphoenolpyruvate via PEPCK which can be used for gluconeogenesis. Acetoacetate can be released as a ketone body or converted to 2 acetyl-CoAs. Both carbons in acetyl-CoA are lost as CO2 in the TCA cycle and therefore acetoacetate can't be used to make glucose.

After fasting for 12 hours, your blood glucose is low. Describe the signal cascades this causes in the liver (glycogenolysis vs. gluconeogenesis), and how this ultimately affects blood glucose levels.

Lower blood glucose leads to glucagon secretion by α-cells in the pancreas. Glucagon raises cAMP levels in cells, which activates PKA, activating phosphorylase b kinase , which phosphorylates glycogen phosphorylase b, converting it to glycogen phosphorylase a. Glycogen phosphorylase a releases glucose 1-phosphate from glycogen, which after being dephosphorylated by glucose-6-phosphatas in the ER, is converted to glucose and released from the liver cell to replenish blood glucose. Glucagon also activates FBPase-2, reducing F2,6Bisphophosphate, which increases FBPase-1 activity, causing glycolysis to slow down and gluconeogenesis to increase.

How are TCA cycle intermediates made accessible for use in the cytoplasmic reactions of gluconeogenesis? How are they most commonly replenished?

Malate is transported from the mitochondrion to the cytoplasm where it is converted to oxaloacetate and subsequently to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. Phosphoenolpyruvate can then be directly used for cytoplasmic gluconeogenesis. Malate transport from mitochondria depletes TCA cycle intermediates which are most commonly replaced by the carbon backbones of glucogenic amino acids.

How is the urea cycle integrated with the TCA cycle?

Mitochondrial OAA is withdrawn from the TCA cycle in this transamination reaction: Glutamate + OAA → α-ketoglutarate + aspartate This asparate is needed for the formation of arginine succinate. The α-Ketoglutarate can resupply the OAA by conversion to succinyl-CoA→succinate→ fumarate→malate→OAA. Fumarate released from argininosuccinate is converted to malate by cytosolic fumarase and transported back into mitochondria where is can be converted back to OAA completing its cycle.

What role does N-acetylglutamate play in the regulation of the Urea cycle in liver cells and which enzyme activity is most directly affected by this molecule?

N-acetylglutamate is an allosteric activator of carbamoyl phosphate synthetase I. High levels of acetylCoA and glutamate indicate that amino acids are being broken down and urea needs to be excreted. The enzyme N-acetylglutamate synthase itself is activated by arginine to sense whether or not there are enough urea cycle intermediates for rapid production of urea.

Briefly describe the role of phosphoprotein phosphatase 1 (PP1) in glycogen metabolism.

PP1 dephosphorylates phosphorylase kinase, glycogen phosphorylase, and glycogen synthase. PP1 is activated by insulin, and the overall effect is to stimulate glycogen synthesis.

List how Glycogen synthase kinase 3 (GSK3) is activated and deactivated by other kinases or small signaling molecules. Also name the protein that it phosphorylates in glycogen metabolism.

Phosphorylation of glycogen synthase by Casein Kinase II (CKII) enhances GSK3 binding. CSK3 needs to be dephosphorylated to be active so PP1 probably activates it as well. It is deactivated by phosphorylation by PKB It phosphorylates glycogen synthase deactivating it

Draw the reaction catalyzed by glycogen phosphorylase.

Pi + Non-reducing terminus of glycogen(n glucose residues) ↔ glucose-1-phosphate + glycogen(n-1 glucose residues)

Which steps in the glycine and L-lactate pathways provide the reducing equivalents to convert 1,3-diphosphoglycerate to glyceraldehyde-3-phosphate during gluconeogenesis? Just name the enzymes that produce NADH.

The NADH required for reversing the glycolytic pathway from pyruvate is provided by the glycine cleavage enzyme. In the case of the L-lactate pathway, the NADH is provided by lactate dehydrogenase for the oxidation of lactate to pyruvate.

You notice that a friend's breath smells like acetone. Name two possible causes of "acetone" breath and suggest how you would distinguish between them

The acetone breath is almost certainly the result of high levels of ketone bodies in the friend's blood. Ketone bodies consist of β- OH butyrate and acetoacetate, and the latter is decarboxylated in the lungs to acetone. The cause for the build up of ketone bodies is probably due to either starvation (due to fasting or anorexia nervosa) or diabetes mellitus. If you friend eats candy bars containing lots of glucose and other sugars, the ketone bodies in the blood and the acetone smell should go away as blood glucose levels rise, TCA cycle intermediates build up, and acetylCoA from fatty acid oxidation becomes completely combusted to CO2 and H2O. If your friend has uncontrolled, type 1 diabetes the ketone body levels will remain high because the ingested glucose cannot be rapidly taken up and converted into TCA cycle intermediates for processing acetylCoA from fatty acid oxidation. ***The simplest test and best answer would be draw blood and look at the blood glucose level. In diabetes it would be high and in starvation it would be low - my answers above are if you had no access to a clinical laboratory ***

What prevents pyruvate kinase from converting the phosphoenolpyruvate (PEP) formed from oxaloacetate by PEP carboxykinase from being converted to pyruvate during gluconeogenesis?

The answer must include a description of the allosteric negative regulators, acetyl- CoA and long chain fatty acids, which occurs in all tissues and the hormonal regulation by glucagon triggered phosphorylation in liver.

Name a G-protein coupled receptor (GPCR) and its agonist (hormone that binds to and activates it), which stimulates glycogen breakdown and lipolysis in adipose tissue even in the presence of insulin. Explain how its signaling pathway can override the effect of insulin in the case of glycogen breakdown.

The best answer is the epinephrine or adrenaline receptor, which is a GPCR that stimulates adenylate kinase producing cAMP. cAMP activates protein kinase A (PKA), which in turn, phosphorylates a number of proteins in the glycogen particle, which lead to activation of glycogen phosphorylase and inactivation of glycogen synthase, even in the presence of insulin. The key phosphorylations are: (1) The Gm protein is phosphorylated a second time which releases protein phosphatase 1 (PP1) from the particle. (2) Inhibitor protein 1 is phosphorylated and binds the released PP1, keeping it out of the particle. (3) Phosphorylase b kinase is activated by phosphorylation by PKA and in turn actives glycogen phosphorylase causing glucose release. *high levels of glucagon and insulin are never present at the same time unless the pancreas is in a severe diseased state*

When rats with pernicious anemia (vitamin B12 deficiency) are fed this oil as their primary source of fats, they become disoriented due the presence of high concentrations of the following compound, which can cross the blood brain barrier and inhibit succinate dehydrogenase in the TCA cycle. Name the compound and explain in detail why it is present in the blood of these rats.

The compound is methylmalonate. It is generated from the breakdown of methylmalonylCoA which is an intermediate in the pathway for the conversion of propionylCoA to succinylCoA . The R3 odd chain fatty acid margaric acid (17:0) in the oil will undergo β-oxidation until propionylCoA is formed. In healthy patients, who have sufficient vitamin B12, this propionylCoA will be converted to succinylCoA by the pathway pictured. The last step, methylmalonylCoA mutase requires cobalamin or vitamin B12. If vitamin B12 is missing this step is blocked and methylmalonylCoA builds up and eventually is hydrolyzed to methylmalonate, which at high concentrations will inhibit succinate dehydrogenase in the TCA cycle.

White adipocytes store and provide unsaturated fatty acids (CoA-conjugated structure shown on reverse) to mitochondria. What is the potential issue with the β-oxidation of this fatty acid? What enzyme overcomes this issue? Explain what the enzyme does?

The conventional β- oxidation reaction cannot take place at the unsaturated site (undesirable C=C double bond at the site of beta oxidation). Enoyl CoA isomerase converts the cis unsaturated bond to the trans form which is a suitable intermediate for β-oxidation.

Assume that the fatty acids are taken up into the cytoplasm of heart myocytes and acylated to CoA derivatives. Then describe how these three fatty acids are transported into mitochondria by describing in words the reactions involved in this transfer. What roles do 4-trimethylamino-3- hydroxybutyrate (carnitine) and malonylCoA play in these processes?

The fatty acyl groups of the acyl-CoA are transferred to the hydroxyl group of carnitine by carnitine acyltransferase I in the outer mitochondrial membrane facing the cytoplasm and then transported through the inner membrane by a protein transporter and then transferred back to CoASH by carnitine acyltransferase II in the mitochondrial matrix. MalonylCoA is an allosteric inhibitor of the outer membrane (cytoplasmic) carnitine acyltransferase I. If it is present, as is the case during fatty acid synthesis in hepatocytes, then no transport of acylCoA groups into mitochondria occurs.

What additional steps are required to utilize odd chain fatty acids in β- oxidation?

The final step in β-oxidation will result in formation of the 3 carbon substrate, propionyl-CoA which must be converted to methymalonyl-CoA by propionyl-CoA carboxylase. Methymalonyl-CoA then must be converted to succinyl-CoA by methylmalonyl mutase whereupon it can be utilized in the TCA cycle.

In liver cell lysates, the conversion of L-lactate to glucose requires the addition of catalytic amounts of bicarbonate, whereas the conversion of glycine to glucose does not. Explain this difference.

The glycine cleavage enzyme produces CO2 when generating THF-CH2OH for transfer to the second glycine. Thus, glycine condensation to make serine produces its own bicarbonate for the pyruvate carboxylase/PEPCK reactions where it is used catalytically. In the case of lactate, bicarbonate has to be added for these reactions to occur.

During strenuous exercise muscle glycogen is depleted and muscle becomes heavily dependent on the liver to supply glucose. It seems that it might be more efficient for muscle to be able to generate its own glucose. Why do you think this doesn't happen?

The need to generate ATP via glycolysis and TCA cycle (aerobic oxidation) is intense during strenuous exercise. Conditions that support glucose breakdown and gluconeogenesis are highly regulated and opposed such that both don't occur at the same time.

On average what is the difference in the net ATP (high energy phosphate) yield per carbon in the complete oxidative catabolism of glucose versus the 16 carbon fatty acid palmitate (16:0).

The net yield for glucose is 30 - 32 ATP = 5 - 5.3 ATP/carbon The net yield for palmitate is 106 ATP (108 ATP are generated but require 2 high energy phosphates to charge fatty acid onto CoA initially) → 106 ATP/16 carbons = 6.6 The difference = 1.3 - 1.6 ATP/carbon

δ-Aminolevulinic acid is a key precursor for the synthesis of tetrapyrroles including all porphyrins and hemes. It is biosynthesized by the condensation of succinyl-CoA with glycine as shown on the other side. From your knowledge of amino acid catabolism, name the cofactor that is required. Then write out a detailed mechanism showing how the cofactor facilitates the reaction.

The reaction involves decarboxylation of glycine and nucleophilic displacement of CoASH by an α-carbanion of glycine to form δ- aminolevulinate. Thus the reaction must involve a pyridoxal phosphate cofactor.

What extra enzyme is needed for the complete β-oxidation of R2, palmitoleic acid 16:1(Δ ) and why?

The Δ3 cis double originally at the 10-9 position has to be isomerized to trans double bond at the 3-2 position to allow β-oxidation to continue (1pt - doesn't need to be this detailed). The enzyme is Δ3Δ2-enoyl-CoA isomerase.

Describe the interrelationships between muscle and liver with regard to amino acid metabolism under anaerobic conditions.

Under anaerobic conditions, alanine, from muscle protein break down, is released into the blood. The liver takes up alanine from blood and converts the nitrogen to urea and the carbons to pyruvate and then glucose, which, in turn, is released back into the blood and for use by muscle and other tissues as a source of energy.

A mutation in the enzyme Phosphofructokinase-2/Fructose bisphosphatase-2 has been discovered that prevents it from being phosphorylated by protein kinase A. What impact would you expect on glycolysis and gluconeogenesis?

Unphosphorylated PFK-2/FBP-2 would lead to chronic activation of the PFK-2 activity and inhibition of the FBP-2 activity leading to chronic elevation of fructose-2,6- bisphosphate which would drive glycolysis and inhibit gluconeogenesis. The main metabolic problem to be expected is a defect in gluconeogenesis.

Protein phosphatase 1 (PP1) plays a key role in glycogen metabolism. When it is bound to the glycogen particle and active, which process is stimulated, glycogen breakdown or glycogen synthesis and why?

When PP1 is bound to glycogen particles and active, it dephosphorylates all of the key enzymes, which results in inactivation of glycogen phosphorylase and activation of glycogen synthase. Both the last two processes lead to net synthesis and storage of blood glucose as the branched polysaccharide.

Name 5 distinct ways in which the level of activity of a metabolic enzyme can be controlled.

- transcription/gene regulation - mRNA stability - protein translation - protein degradation - substrate availability - allosteric regulation - phosphorylation/dephosphorylation - association with regulatory protein(s) - sequestering/compartmentalization

In liver cells what are the key steps that remove the amino groups from glutamine for use in the urea cycle and the energetic (high energy phosphate equivalents) cost to convert these amino groups to urea? Indicate at which steps in this process where the high energy phosphate equivalents are used.

1 amino group is removed as NH3 by glutaminase and the amino group of the resulting glutamate is removed by transamination of OAA via glutamate dehydrogenase creating α-ketoglutarate aspartate. The released NH3 is converted to carbamoyl phosphate by carbamoyl synthetase I requiring 2 ATPs (2 high energy phosphate equivalents. Citrulline condenses with ATP and forms argininosuccinate at the cost of two more high energy phosphate equivalents since PPi is released which is further broken down to 2 Pi.

How many ATP equivalents (high energy phosphate bonds) are needed to generate UDP-glucose from glucose? Indicate the steps where these are used in the process.

1 from Hexokinase conversion of glucose to glucose-6-phosphate and 2 more in the conversion of glucose-1-phosphate + UTP to UDP-glucose + PPi (the PPi is then hydrolyzed by pyrophosphatase to 2 Pi) → a total of 3 high energy phosphate bonds.

Name the steps in glycolysis that are irreversible and describe how they are overcome in the process of gluconeogenesis. Include any key regulators of the steps as appropriate.

1. Hexokinase Glucose-6-phosphate is transported into the endoplasmic reticulum (ER) and the dephosphorylated by glucose-6-phosphatase. Glucose and phosphate released by this reaction are transported back to the cytoplasm. Glucose + ATP→Glucose-6-phosphate + ADP 2. Phosphofructokinase 1 (PFK-1) Reversed by the action of fructose- bisphosphatase-1 (FBP-1). PFK-1 is stimulated and FBP-1 is inhibited by fructose- 2,6-bisphosphate. Fructose-2,6-bisphosphate levels are controlled by the activity of PFK-2/FBP-2, a single protein with 2 both activities. Fructose-6-phosphate + ATP → Fructose-1,6-bisphosphate + ADP 3. Pyruvate kinase (PK) Phosphoenolpyruvate + ADP → Pyruvate + ATP This is reversed through the action of phosphoenolpyruvate (PEP) carboxykinase (CK) using either: 1)oxaloacetate from the mitochondria reduced/converted to malate in the mitochondrion, transported via the malate shuttle to the cytoplasm, reoxidation to OAA generating NADH in the cytoplasm, with loss of CO2 via cytoplasmic PEP CK and generation of PEP or; 2) lactate via oxidation/conversion to pyruvate in the cytoplasm with NADH generation, transport of pyruvate to the mitochondrion, conversion to OAA and then PEP in the mitochondrion and PEP transport back to the cytoplasm.

Describe the pathway by which fatty acids are mobilized from adipocytes, move from the bloodstream into mitochondria for utilization in β-oxidation. Describe key steps, cellular compartments or organelles involved and key proteins or cofactors mediating this overall process.

1. activation of lipases to release fatty acids from adipocyte lipid droplets 2. transport through bloodstream bound to serum albumin 3. uptake by liver and cytosolic condensation with CoA to form fatty acyl- CoA 4. transfer to carnitine to form fatty acyl - carnitine with release of CoA and transport into mitochondrion 5. transfer from fatty acyl - carnitine to CoA to form fatty acyl - CoA in mitochondrion

Describe the pathway by which fatty acids from dietary fats are digested and absorbed by the digestive tract, repackaged and transported to tissues. Name the proteins involved and describe their functions.

1. emulsification by bile salts in intestine 2. release of fatty acids by intestinal lipases 3. absorption by intestinal epithelia and repackaging into chylomicrons 4. chylomicron transport through blood and lymphatics 5. fatty acid release from chylomicrons by lipases (clearing factor) and uptake and utilization by tissues

A newly discovered marine organism (Vibrio periergo) can feed on 3-phosphoglycerate (3-PG) directly derived and transported from its environment. It has an intact glycolytic system and TCA cycle. What is the net ATP (high energy phosphate) yield from the complete glycolytic and oxidation of one molecule of 3-PG?

3-PG generates 1 ATP during conversion to pyruvate Pyruvate generates: 4 NADH (x 2.5 ATP/NADH = 10 ATP) 1 FADH2 (x 1.5 ATP/FADH2) 1 GTP (= 1 ATP equivalent) during oxidation in the TCA cycle = 10 + 1.5 + 1 = 12.5 Total = 13.5 ATP (high energy phosphate) equivalents