L13: Acetyl-CoA and TCA Cycle

Question 1

What does a low CHO diet cause?

Answer 1

• Low CHO diet leads to ketosis only

Question 1

What does type 1 or 2 diabetes cause?

Answer 1

• Ketosis and ketoacidosis can occur primarily in uncontrolled type 1 diabetes and in some type 2 diabetics.

Question 2

PDH complex. a.) Describe its structure? b.) Where is it located? What is its function? c.) What are its coenzymes, vitamins? d.) What are its substrates and products? Show the balanced reaction it catalyzes. e.) Explain mechanisms for regulation of PDH complex. Explain when it is active and inactive.

Answer 2

• a.) PDH is a 3 enzyme complex. It contains a pyruvate decarboxylase (aka pyruvate dehydrogenase), a dihydrolipoyl transacetylase and a dihydrolipoyl dehydrogenase - b.) It is located in the mitochondrial matrix and serves to link glycolysis to the TCA cycle - c.) It requires 5 coenzymes: TPP, FAD/FADH2, NAD/NADH2, CoASH and lipoamide and 4 B-vitamins: thiamin (e) – B1, riboflavin – B2, niacin – B3 and pantothenic acid – B5. - d.) CH3COCOO- + NAD+ + CoASH = CH3COSCoA + CO2 + NADH + H+. The reaction is not reversible as the delta Go’ is too high. Too much free energy is lost. - e.) PDH is inactive when phosphorylated and is therefore activated by PDH phosphatase. PDH is deactivated when phosphorylated by PDH kinase. It is activated during times when ATP energy is needed: low concentrations of CoASH, pyruvate, ADP and NAD+ (substrates of reaction). These states deactivate the kinase, leading to active form of PDH. It is deactivated during times when ATP energy is not required: high concentrations of acetyl-CoA and NADH (products of reaction). These states activate the kinase, leading to inactive form of PDH.

Question 2

What are causes of excessive acetyl-CoA production?

Answer 2

• starvation, low CHO diet and type I and 2 diabetes

Question 3

Acetyl-CoA. a.) What is its structure? b.) What is its metabolic importance? c.) What is its source? Where does it come from?

Answer 3

• a.) see picture - b.) It is an important intermediate and precursor molecule for TCA, FA biosynthesis, ketone body formation and cholesterol biosynthesis (leading to steroid hormones, bile acids and vit D production) - c.) It is produced as a result of glycolysis, beta-oxidation and AA deamination/oxidation. It is requires presence of vitamin B5.

Question 3

What biosynthetic precursors is the TCA cycle a source of?

Answer 3

• Amino acids, urea, purines, pyrimidines, glucose, fatty acids, sterols, porphyrins

Question 4

What is ketosis and ketoacidosis? What causes these conditions?

Answer 4

• Ketosis refers to the increased production of ketone bodies that doesn’t produce an acidemia - Ketoacidosis refers to increased production of ketone bodies that are high enough to lower the blood pH and produce an acidemia - They are caused by excessive acetyl-CoA production which occurs in uncontrolled type 1 diabetics and in some type 2 diabetics.

Question 5

What does starvation cause?

Answer 5

• Starvation leads to ketosis only

Question 6

Pyruvate carboxylase. a.) What is the function of this reaction? b.) Write out the reaction is catalyzes. Include all energy components and coenzymes. c.) What is an allosteric activator of this enzyme? Why is that important?

Answer 6

• It is an anaplerotic reaction to regenerate oxaloacetate when levels are low. - Pyruvate + ATP + HCO3 = oxaloacetate + ADP + Pi + H+ (ez: pyruvate carboxylase, coenzyme: biotin) - Acetyl-CoA. When there is sufficient amount of acetyl-CoA and therefore little need to divert more pyruvate into acetyl-CoA, pyruvate carboxylase converts pyruvate to oxaloacetate.

Question 8

What are the consequences of PDH deficiency? Population affected? Symptoms?

Answer 8

• Leads to chronic lactic acidosis, primary in kids. It leads to severe neurological problems and is frequenctly fatal.

Question 9

Thiamin deficiency. a.) What vitamin is deficient? Where is this required? b.) Who does it affect? c.) What are the early symptoms? d.) What are the advanced / severe deficiencies of thiamin? Which groups are affected? What are the symptoms?

Answer 9

• a.) Vitamin B1. Required for PDH and alpha-KGDH enzymes to function. - b.) Primarily affects alcoholics, elderly and low income groups who are more likely to have poor diets. - c.) loss of appetite, constipation, nausea, fatigue, depression, peripheral neuropathy - d.) Wernicke-Korsakoff = advanced deficiency frequently seen in alcoholics: mental confusion, ataxia, loss of eye coordination Beri-Beri = severe deficiency seen in populations that heavily rely on polished rice diets: neuromuscular symptoms – muscle weakness, muscle atrophy, fatigue, peripheral neuropathy, lactic acidemia

Question 11

What enzyme(s) require(s) thiamin (B1)?

Answer 11

• Alpha-ketoglutarate dehydrogenase complex and PDH complex.

Question 12

What is an anaplerotic reaction? What is the importance of these reactions for the TCA cycle? What is an important anaplerotic reaction for the TCA cycle? Include enzyme and coenzymes required. Are there other reactions?

Answer 12

• Anaplerotic reactions are those that replenish substrates. The TCA cycle is not an end-product producing reaction. It re-generates oxaloacetate in order for the cycle to occur again. However; TCA cycle intermediates, including oxaloacetate, are constantly being drawn away from the cycle as they serve as intermediates for biosynthetic pathways and therefore the oxaloacetate concentrations decrease preventing expanding rates of the TCA cycle should the need occur. - Pyruvate + ATP + HCO3 = oxaloacetate + ADP + Pi + H+ (ez: pyruvate carboxylase, coenzyme: biotin) - Yes, any reaction that generates an intermediate of the TCA cycle is a potential anaplerotic reaction. There is a reaction involving glutamate and glutamate dehydrogenase that generates more alpha-ketoglutarate, which adds to oxaloacetate in subsequent reactions.

Question 13

Name and draw the ketone bodies produced in the body.

Answer 13

Question 14

What enzyme deficiencies can lead to lactic acidosis?

Answer 14

• PDH, pyruvate carboxylase, any of the CAC enzyme defects.

Question 15

What is Wernicke-Korsakoff syndrome? What are the symptoms?

Answer 15

• It is an advanced thiamin (B1) deficiency that typically occurs in alcoholics. Symptoms include mental confusion, ataxia and loss of eye coordination.

Question 16

Pyruvate. a.) What is the fate of pyruvate? b.) What enzyme converts pyruvate to acetyl-CoA? c.) What enzyme converts pyruvate to oxaloacetate? d.) What determines whether pyruvate is used to form acetyl-CoA or oxaloacetate?

Answer 16

• a.) lactate, oxaloacetate, acetyl-CoA and alanine - b.) PDH complex - c.) pyruvate carboxylase - d.) pyruvate carboxylase is allosterically activated by acetyl-CoA. When there is sufficient amount of acetyl-CoA and therefore little need to divert more pyruvate into acetyl-CoA, pyruvate carboxylase converts pyruvate to oxaloacetate.

Question 17

What is biotin? Which vitamin is it?

Answer 17

• Biotin is a B vitamin, also known as vitamine B7. It is required by pyruvate carboxylase.

Question 18

Beri-beri. a.) What is it? What is it caused by? b.) What are the symptoms associated with it? c.) What are the two forms of this disorder? d.) What organ/tissue(s) are most affected by this disorder? e.) Do RBCs contribute to this condition? Explain. f.) Which populations are susceptible to this disorder?

Answer 18

• a.) It is a severe thiamin (B1) dietary deficiency. - b.) Neuromuscular symptoms – muscle weakness, muscle atrophy, fatigue, peripheral neuropathy and lactic acidemia. - c.) Dry beri-beri which affects neuromuscular function, does not lead to fluid retention; Wet beri-beri causes peripheral edema and cardiac failure. - d.) It affects the nervous system and musculoskeletal system primarily. - e.) No, as they do not contain mitochondria, where enzymes such as PDH complex and aKGDH that participate in linking glycolysis to TCA cycle and TCA cycle function respectively. - f.) Populations that have high intake of polished rice diets.

Question 20

TCA cycle. a.) What are the 2 major functions of the TCA cycle? b.) What is the overall balanced reaction of the TCA cycle? c.) Draw the reactions of the TCA cycle. Include enzymes, cofactors. d.) What is the fuel for the TCA cycle? e.) Which reaction(s) liberate CO2? f.) Which reaction(s) liberate NADH? g.) Which reaction(s) liberate FADH2? h.) Which reaction(s) involve substrate-level phosphorylation? i.) How many ATPs/ATP-equivalents are produced from a molecule of acetyl Co-A? From pyruvate? From glucose? j.) Explain mechanisms for TCA cycle regulation. What enzymes are regulated?

Answer 20

• a.) Serves to generate ATP and to generate biosynthetic precursors. - b.) CH3COSCoA + 2H2O + 3NAD+ + FAD + GDP + Pi = 2CO2 + 3NADH + 3H+ + FADH2 + CoASH + GTP - c.) see picture - d.) Acetyl-CoA - e.) isocitrate to alpha-ketoglutarate; alpha-ketoglutarate to succinyl-CoA - f.) isocitrate to alpha-ketoglutarate; alpha-ketoglutarate to succinyl-CoA; malate to oxaloacetate - g.) succinate to fumarate - h.) succinyl-CoA to succinate - i.) since 3 ATPS per NADH and 2 ATPS per FADH - 12 per acetyl CoA, 15 from pyruvate (considering PDH reaction) and 30 from glucose - j.) Regulation of the TCA cycle occurs through regulation of isocitrate dehydrogenase and alpha-ketoglutarase dehydrogenase complex. Isocitrate dehydrogenase activity increases when conc of ADP are high and decreases when NADH conc are high. Alpha-ketoglutarate dehydrogenase complex activity decreases when concentrations of succinyl-CoA and NADH are high.

Question 21

How is the PDH complex similar or dissimilar to the alpha-ketoglutarate DH complex?

Answer 21

• Each complex has 3 enzyme subunits. Each produces CO2. Substrate for PDH is 3C alpha-keto acid (pyruvate), while substrate for aKGDH is 5C alpha-keto acid (alpha-ketoglutarate). Product for PDH is 2C (acetyl-CoA), while produce for aKGHD is 4C (succinyl-CoA). Regulation of PDH occurs via phosphorylation, while aKGDH is not regulated via phosphorylation. Reactions catalyzed by both are irreversible as a lot of free energy is lost.

Question 22

Besides pyruvate, what are other sources of acetyl CoA? What happens when too much acetyl-CoA is produced by the liver? When are cases when this can occur? Is this detrimental? Is this beneficial?

Answer 22

• Glucose via pyruvate, FFAs via beta-oxidation and amino acids via deamination/oxidation - Too much acetyl-CoA causes formation of ketone bodies. - Can occur during fasting, starvation and in uncontrolled type 1 diabetics (sometimes in type 2 diabetics). - Production when moderate is generally a great benefit in the body, allowing it to withstand nutrient deprivation. It is especially useful in brain as the brain cannot use FA stores from body. When production is too great; however, acid environment forms (ketoacidosis) leading to coma and death, mostly in type 1 diabetics.