CAC Focus

Write the overall reaction catalyzed by the pyruvate dehydrogenase (PDH) complex and define oxidative decarboxylation.

pyruvate + CoA + NAD+ ←> acetyl coA + CO2 + NADH + H+

• Oxidative decarboxylation: irreversible oxidation- carboxyl removed as CO2

• Pyruvate converted to acetyl-CoA, irreversible, so commits pyruvate carbon to oxidation/uses for acetyl-coA

Name the five cofactors used by the PDH complex

NAD+, TPP, lipoate, FAD, coenzyme A

Why it is advantageous for the PDH complex to channel its intermediates between active sites rather than releasing them.

Passes intermediates from 1 active site to another in 5 reactions

• channeling speeds up overall rxn since intermediates don’t need to diffuse bw enzymes, keeps intermediates from being lost

Compare the PDH complex with the α-ketoglutarate dehydrogenase complex, and explain what their similarities suggest about how these enzymes evolved

PDH complex: oxidative decarboxylation where pyruvate → acetyl coA

a keto-glutarate dehydrogenase complex: oxidative decarboxylation akg →succinyl coA

• Both: 5 cofactors— Coenzyme A, NAD+, FAD, TPP, lipoate and release CO2

• Similar E1 and E2, identical E3

• Suggest duplication → divergent evolution

Starting with acetyl-CoA and oxaloacetate, list the eight reactions of the citric acid cycle in order, naming each enzyme and its product.

acetyl coA + oxaloacetate + H2O → citrate + SH-COA enzyme: citrate synthase

citrate + H2O ←> cis aconitate ←> isocitrate + H2O enzyme: aconitase

isocitrate + NAD+ ←> a ketoglutarate + NADH + CO2 isocitrate dehydrogenase

a ketoglutarate + NAD+ + HS-CoA ←> succinyl coA + NADH+ H+ +CO2

• a ketoglutarate complex: coenzyme A, NAD+, TPP, lipoate, FAD

succinyl coA + GDP ←> succinate + HS-CoA + GTP enzyme: succinyl coA synthetase

succinate + FAD ←> fumarate + FADH2 enzyme: succinate dehydrogenase

fumarate + OH- + H+ ←> L malate enzyme: fumarase

malate + NAD+ ←> oxaloacetate + NADH enzyme: malate dehydrogenase

reaction for oxaloacetate + acetyl coA →citrate, enzyme

oxaloacetate + acetyl coA + H2O → citrate + HS-CoA

• citrate synthase

Reaction for citrate → isocitrate

citrate + H2O ←> cis aconitate ←> isocitrate + H2O

• aconitase

Reaction for isocitrate → a-ketoglutarate

isocitrate + NAD+ → a-ketoglutarate + NADH + CO2

• isocitrate dehydrogenase

Reaction for a-ketoglutarate to succinyl coA

a-ketoglutarate + HS-CoA + NAD+ → succinyl coA + CO2 + NADH

• cofactors: coenzyme A, NAD+, FAD, TPP, lipoate

• enzyme: a-ketoglutarate dehydrogenase complex

Reaction for succinyl coA to succinate

succinyl coA + GDP ←> succinate + GTP + HS-CoA

• succinate dehydrogenase

What enzyme converts GTP to ATP

GTP + ADP ←> GDP + ATP

• nucleoside diphosphate kinase

Reaction for succinate to fumarate

succinate + FAD ←> fumarate + FADH2

Reaction for fumarate to malate

fumarate + H2O ←> malate

• fumarase

reaction for malate → oxaloacetate

malate + NAD+ ←> oxaloacetate + NADH + H+

• malate dehydrogenase

• pulled forward by low oxaloacetate

Identify the four oxidation steps and the electron carrier reduced in each, and the two steps that release CO₂.

oxidations: steps 3,4,6,8

• step 3,4,8 : NAD+ step 6: FAD

decarboxylations: steps 3,4

• succinate dehydrogenase= intergral protein of mito w cov. bound FAD, iron cluste

malate dehydrogenase reaction has a positive ∆G′°. Explain how the cycle still moves forward, and why the large negative ∆G′° of citrate synthase is needed

• oxaloacetate usually kept at very low conc, makes ∆G for malate dehydrogenase negative even though ∆G′° is positive

• Since oxaloacetate so scarce, citrate needs large negative ∆G′° to condense efficiently

  • similarly, low isocitrate pulls aconitase reaction forward

Explain how succinyl-CoA synthetase captures energy, and the role of nucleoside diphosphate kinase.

Breaks the high-energy thioester bond of succinyl-CoA, uses energy to make GTP or ATP

• nucleoside diphosphate kinase interconverts GTP and ATP —>net result=ATP, no matter which phosphorylated

List the energy-carrying products of one turn of the citric acid cycle. Explain why the cycle directly generates only one ATP per turn, but is still the major source of ATP in aerobic cells.

1 turn: 3 NADH, 1 FADH2, 1 GTP

• directly generates 1 ATP/turn, but converts to 10 ATP/turn when carries oxidized in ETC

calculate the ATP yield from one acetyl-CoA oxidized by the cycle, and from one pyruvate

oxidized through the PDH complex and the cycle

1 acetyl coA oxidized: 3 NADH, 1 FADH2, 1 GTP -→ 10 ATP

1 pyruvate: +1 NADH, so 12.5 ATP

Explain how the complete aerobic oxidation of one glucose yields 30 to 32 ATP, and why the total is given as a range

In glycolysis: net 2 ATP, 2 NADH (3 or 5 ATP) 2 pyruvate

2 pyruvate → 2 acetyl coA + 2 NADH + 2 x CAC (3 NADH, 1 FADH, 1 GTP)

• give 8 NADH, 2 FADH, 2 ATP = 25 ATP from PDH and CAC

The total is 2 ATP + (3 or 5) + 25 = 30 to 32 ATP

• Range from the cytosolic NADH made in glycolysis. It cannot enter mitochondria directly, and depending on which shuttle carries its electrons inside, each yields about 1.5 or 2.5 ATP

Define amphibolic, and explain how the citric acid cycle serves in both catabolism and anabolism

Amphibolic: catabolic and anabolic pathways

CAC oxidizes acetyl-CoA for energy and intermediates are used as precursors for biosynth

• oxaloacetate for glucose, aspartate

• a-ketoglutarate for glutamate

• citrate for fatty acids

Give examples of amino acids that feed carbon skeletons into the cycle.

Cycle also accepts 3-, 4-, 5-carbon skeletons from AA breakdown

• deaminated aspartate → oxaloacetate

• deaminated glutamate → a ketoglutarate

Explain why animals cannot make net glucose from acetyl-CoA, and how organisms with the glyoxylate cycle can

Animals cannot convert acetate or acetyl-CoA to glucose

• in CAC, each acetyl-CoA brings 2 carbons in, 2 carbons leave as CO2

• no net conversion of acetate → oxaloacetate, so acetyl coA can’t supply net carbon for gluconeogenesis

• PDH irreversible, so can’t turn acetyl-coA back to pyruvate

Glyoxylate cycle: converts acetate to carbohydrate— bacteria, plants, fungi, protists

• bypasses the two decarboxylation steps, allows net conversion of acetate to 4-C intermediates, then to carbs

Define anaplerotic reactions and explain why they are needed

Replenish citric acid cycle intermediates that are withdrawn for biosynthesis. Without them, the cycle would run down and could not oxidize acetyl-CoA.

Describe the pyruvate carboxylase reaction and explain why it makes sense that acetyl-CoA activates this enzyme.

pyruvate + HCO3- + ATP → oxaloacetate + CO2 + ADP + Pi

• The enzyme requires biotin and is allosterically activated by acetyl-CoA.

• Accumulating acetyl- CoA = not enough oxaloacetate to condense with it, so activating pyruvate carboxylase supplies more

Identify the four regulated steps

PDH complex

citrate synthase

isocitrate dehydrogenase

a-ketoglutarate dehydrogenase complex

High [ATP]/[ADP] and [NADH]/[NAD+] mean energy plentiful, steps inhibited

• ADP, NAD+ signal high demand → activate

• Ca2+ released when muscle contracts, activate isocitrate dehydrogenase, a ketoglutarate complex, PDH complex

How is PDH complex regulated

Allosterically, PDH complex inhibited by products (ATP, NADH, acetyl coA, FA) and activated by substrates (AMP, CoA, NAD+)

PDH (inactive) → PDH (active) with PDH kinase

• activated by products ATP, NADH, acetyl coA, Ca2+

• inhibited by substrates ADP, NAD+, pyruvate, FA

PDH (active) → PDH (inactive) w PDH phosphatase

How is citrate synthase regulated

Activated: ADP

inhibited: NADH, succinyl-coA, citrate, ATP

How is isocitrate dehydrogenase regulated

Activated: Ca2+, ADP

Inactivated: ATP

How is a ketoglutarate complex regulated

Activated: Ca2+

Inactivated: succinyl coA, NADH

Person fasting, oxidizes fatty acids, so higher acetyl-CoA, NADH, ATP in liver mito

• Predict effect on PDH complex, pyruvate carboxylase

Don’t need acetyl coA, NADH, ATP —>need to inactivate PDH

• FA, acetyl-coA, NADH, ATP activates PDH kinase —>inactivates PDH

Need to replenish glucose, so pyruvate carboxylase activated for gluconeogenesis

• pyruvate from sources like lactate, alanine is saved for making glucose while FA supply energy

Predict and explain how a working skeletal muscle cell adjusts flux through the cycle.

In working muscle: consume ATP, increase ADP and AMP, reoxidize NADH →NAD+

• Ca2+ released in contraction, activates PDH, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase

• Inc flux through PDH complex, raises ATP production to match demand