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Biochemistry · Metabolism

Citric acid cycle practice: Krebs cycle steps, enzymes, and ATP yield

The citric acid cycle, also called the Krebs cycle or TCA cycle, accepts a 2-carbon acetyl group and releases 2 CO₂ per turn. The incoming carbons stay in the cycle during their first turn and leave in later turns. Almost none of that energy becomes ATP on the spot. It gets loaded onto electron carriers instead: 3 NADH and 1 FADH₂ per turn, plus 1 GTP (or ATP). Learn that scorecard and most exam questions on the cycle become counting problems.

Short on time? Jump to the 10 practice questions, the ATP yield calculator, or the ordering exercise.

The cycle at a glance

Think of oxaloacetate as a 4-carbon carrier. It picks up 2 carbons, the 6-carbon molecule loses 2 carbons as CO₂, and the rest of the ring rebuilds oxaloacetate so it can go again. Because the carrier comes back, a small amount of it can process a lot of acetyl-CoA.

The citric acid cycle as a ring of eight moleculesAcetyl-CoA enters at the top and joins oxaloacetate to make citrate. Going clockwise: citrate, isocitrate, alpha-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, and back to oxaloacetate. NADH and carbon dioxide leave at the two steps after isocitrate and alpha-ketoglutarate, GTP leaves after succinyl-CoA, FAD is reduced after succinate, and NADH leaves after malate.Acetyl-CoA (2C) enters hereOxaloacetate4 carbonsCitrate6 carbonsIsocitrate6 carbonsα-Ketoglutarate5 carbonsSuccinyl-CoA4 carbonsSuccinate4 carbonsFumarate4 carbonsMalate4 carbonsNADHCO₂NADHCO₂GTPFADH₂NADHOne turn2C in, 2 CO₂ out
Eight molecules, eight steps, clockwise from oxaloacetate. The same ring, in words: oxaloacetate, citrate, isocitrate, α-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, then oxaloacetate again.

Acetyl-CoA + 3 NAD⁺ + FAD + GDP + Pᵢ + 2 H₂O → 2 CO₂ + 3 NADH + FADH₂ + GTP + 2 H⁺ + CoA-SH

This guide follows the standard oxidative cycle in mammalian cells. Seven enzymes work in the mitochondrial matrix; succinate dehydrogenase is embedded in the inner membrane. In bacteria, the soluble reactions occur in the cytoplasm and succinate dehydrogenase is membrane-bound. Some microbes use different versions of the pathway. In our mitochondria, no cycle step uses O₂ directly, but sustained oxidation depends on the respiratory chain passing electrons to oxygen.

The 8 steps and enzymes

You do not have to learn these as 8 random facts. The first four steps add 2 carbons and then lose 2 carbons. The last four rebuild the carrier.

The 8 steps of the citric acid cycle, for one acetyl-CoA
StepReactionEnzymeWhat happensCarriers, phosphate, and CO₂ made
1Oxaloacetate + acetyl-CoA → citrateCitrate synthaseCondensation. A 2-carbon acetyl group joins a 4-carbon acceptor to make a 6-carbon acid. CoA is released.Nothing yet
2Citrate → isocitrateAconitaseIsomerization. The OH group moves to a carbon where it can be oxidized.Nothing
3Isocitrate → α-ketoglutarateIsocitrate dehydrogenaseOxidation, then loss of CO₂. 6 carbons become 5.1 NADH, 1 CO₂
4α-Ketoglutarate → succinyl-CoAα-Ketoglutarate dehydrogenase complexOxidation, loss of CO₂, and CoA added. 5 carbons become 4.1 NADH, 1 CO₂
5Succinyl-CoA → succinateSuccinyl-CoA synthetaseThe energy in the thioester bond makes a phosphate bond. This is the only substrate-level phosphorylation in the cycle.1 GTP (ATP in some tissues)
6Succinate → fumarateSuccinate dehydrogenase (Complex II)Oxidation that forms a C=C double bond. This enzyme sits in the inner membrane.1 FADH₂
7Fumarate → malateFumaraseWater adds across the double bond.Nothing
8Malate → oxaloacetateMalate dehydrogenaseOxidation of an OH to a ketone. The acceptor is back, ready for the next turn.1 NADH

To remember the order of the molecules: "Citrate Is Krebs' Special Substrate For Making Oxaloacetate." The K stands for α-ketoglutarate, and the two S words are succinyl-CoA, then succinate.

To remember the enzymes: most are named for what they act on. A dehydrogenase removes hydrogens, so it fills an electron carrier. The cycle has four: isocitrate, α-ketoglutarate, succinate, and malate. That is your 3 NADH plus 1 FADH₂. The odd one out is succinate dehydrogenase. The succinate-to-fumarate reaction cannot favorably reduce NAD⁺, so it uses enzyme-bound FAD. That FAD stays attached to Complex II and passes its electrons to coenzyme Q. The scorecard’s “1 FADH₂” is shorthand for those electrons, not a free FADH₂ molecule leaving the enzyme.

Counting the yield

Almost every yield question is the same three moves: find how many turns you have, multiply the scorecard, and convert to ATP if asked. One glucose gives 2 pyruvate, and each pyruvate gives 1 acetyl-CoA, so one glucose means two turns.

The full ledger for one glucose
StageATP or GTP directlyNADHFADH₂CO₂
Glycolysis2 (net)200
Pyruvate dehydrogenase (2 pyruvate)0202
Citric acid cycle (2 turns)2624
Total41026

Why your textbook may say 30, 32, 36, or 38. Older books count 3 ATP per NADH and 2 per FADH₂. Newer ones use approximate mitochondrial yields of 2.5 and 1.5. These are accounting estimates; actual ATP yield varies with coupling and cell conditions. On top of that, NADH made in the cytoplasm has to get its electrons into the mitochondrion. The malate-aspartate shuttle regenerates NADH in the matrix. The glycerol-3-phosphate shuttle transfers electrons through enzyme-bound FAD to coenzyme Q, bypassing Complex I, so they earn less. Use whatever your instructor uses, and show your values on the exam.

ATP totals under each set of values
Starting fromCurrent values (2.5 and 1.5)Older values (3 and 2)
1 acetyl-CoA1012
1 pyruvate12.515
1 glucose, glycerol-3-phosphate shuttle3036
1 glucose, malate-aspartate shuttle3238

ATP yield calculator

Use this to check your own counting. Work the problem by hand first, then see if you match.

Regulation: read the energy signal

You can answer most regulation questions without memorizing a list. Ask one thing: does this signal say the cell has plenty of energy, or that it needs more? ATP and NADH mean plenty, so the cycle slows. ADP and NAD⁺ mean the cell needs more, so it speeds up. In muscle, calcium signals contraction, so ATP is about to be needed.

Where the cycle is controlled
EnzymeSlowed bySped up by
Pyruvate dehydrogenase (the gate, just before the cycle)Acetyl-CoA, NADH, ATP, phosphorylation by its kinasePyruvate, NAD⁺, Ca²⁺ (through its phosphatase)
Citrate synthase (step 1)Citrate, succinyl-CoA, and high NADH, which also leaves less oxaloacetate. Some textbooks list ATP too.More acetyl-CoA and oxaloacetate
Isocitrate dehydrogenase (step 3)ATP, NADHADP, Ca²⁺
α-Ketoglutarate dehydrogenase (step 4)Succinyl-CoA, NADH, ATPCa²⁺

Those three steps inside the cycle are the ones with large negative free-energy changes, so they are effectively one-way. Isocitrate dehydrogenase is a commonly taught rate-limiting step, though control of flux is shared and depends on conditions.

Step 8 is the surprise. Malate dehydrogenase is uphill, at about +30 kJ/mol under biochemical standard conditions (ΔG°′, pH 7). It still runs forward because citrate synthase uses up oxaloacetate right away, which keeps its level very low.

The cycle also talks to glycolysis. When citrate builds up, some leaves the mitochondrion and slows PFK-1, the main control point covered in the glycolysis quiz.

It is not only for burning fuel. Cells borrow intermediates to build things: citrate for fatty acids, α-ketoglutarate for glutamate, oxaloacetate for aspartate and glucose, and succinyl-CoA for heme. Reactions that refill the cycle are called anaplerotic. The main one is pyruvate carboxylase, which turns pyruvate into oxaloacetate.

Worked examples

Example 1: ATP from one pyruvate

How many ATP equivalents come from fully oxidizing one pyruvate? Use 2.5 per NADH and 1.5 per FADH₂.

  1. Pyruvate dehydrogenase: pyruvate → acetyl-CoA makes 1 NADH and 1 CO₂.
  2. One turn of the cycle makes 3 NADH, 1 FADH₂, and 1 GTP.
  3. NADH: 1 + 3 = 4, and 4 × 2.5 = 10 ATP.
  4. FADH₂: 1 × 1.5 = 1.5 ATP.
  5. GTP: 1 ATP equivalent.
  6. Total: 10 + 1.5 + 1 = 12.5.

Answer: about 12.5 ATP equivalents and a net 3 CO₂. This is net accounting: two of the CO₂ released in that first cycle turn come from the pre-existing oxaloacetate, not the new pyruvate.

Example 2: A whole glucose, using a shuttle

Assume a cell uses the glycerol-3-phosphate shuttle for its glycolytic NADH. Find the ATP from one glucose, using current values.

  1. Direct ATP or GTP: 2 from glycolysis + 2 from the cycle = 4.
  2. Mitochondrial NADH: 2 from pyruvate dehydrogenase + 6 from the cycle = 8, and 8 × 2.5 = 20.
  3. FADH₂: 2 × 1.5 = 3.
  4. The 2 NADH from glycolysis pass their electrons to FAD through the shuttle, so they earn 1.5 each: 2 × 1.5 = 3.
  5. Total: 4 + 20 + 3 + 3 = 30.

Answer: about 30 ATP. With the malate-aspartate shuttle, step 4 would be 5, for a total of 32.

Example 3: Follow a labeled carbon

In a tracing experiment, doubly ¹⁴C-labeled acetyl-CoA enters a cycle whose oxaloacetate starts unlabeled. Do the 2 CO₂ released in the first turn carry the label?

  1. Step 1 attaches the labeled acetyl group to oxaloacetate to make citrate.
  2. The carbons that leave at steps 3 and 4 are carboxyl groups that came from oxaloacetate, not the new acetyl group.
  3. So both labeled carbons are still in the ring at the end of the turn.
  4. Succinate is symmetrical, so after step 5 the enzymes cannot tell its two ends apart. Across many molecules, the labels appear at equivalent positions in regenerated oxaloacetate and leave as CO₂ in later turns.

Answer: No. The CO₂ from the first turn is unlabeled, even though the cycle lost 2 carbons. The carbons lost are not the ones that just came in.

Example 4: Predict what a poison does

Malonate resembles succinate and competitively inhibits succinate dehydrogenase. Predict the immediate trends when it strongly inhibits this enzyme. For this simplified pathway model, ignore other reactions that replenish intermediates.

  1. Malonate competes with succinate at succinate dehydrogenase’s active site. Similarity alone does not prove an inhibitor’s mechanism. See competitive vs noncompetitive inhibition.
  2. Less succinate is consumed, so it tends to accumulate while upstream reactions continue.
  3. Less fumarate is supplied, so fumarate, malate, and oxaloacetate tend to fall in this simplified model.
  4. Less oxaloacetate means citrate synthase has less to work with, so the entire cycle slows.
  5. Because the inhibition is competitive, adding a lot more succinate can partly overcome it.

Answer: succinate tends to build up, downstream supply and electron transfer through Complex II decrease, and the cycle slows. The extent depends on inhibitor and substrate concentrations; competitive inhibition does not automatically stop the enzyme completely.

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Put the intermediates in order

These 8 molecules are scrambled. Sort them into cycle order, with citrate at the top, ending with oxaloacetate. Use the Up and Down buttons, then press Check order.

  1. Succinate
  2. Citrate
  3. Malate
  4. α-Ketoglutarate
  5. Oxaloacetate
  6. Isocitrate
  7. Fumarate
  8. Succinyl-CoA

Show the correct order

Citrate, isocitrate, α-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, oxaloacetate.

Practice questions

These mix multiple choice, select-all, and type-a-number questions, since exams use all three. A wrong answer tells you what went wrong so you can try again, and every question has a full solution.

Question 1Where in a eukaryotic cell does the citric acid cycle run, and which enzyme is the exception?

Show solution

The cycle runs in the mitochondrial matrix. Succinate dehydrogenase is bound to the inner membrane, which lets it hand electrons from FADH₂ straight to the electron transport chain.

Question 2Which enzymes make NADH? Select all that apply.

Show solution

Isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase each make 1 NADH per turn. Four enzymes in the cycle are dehydrogenases; the fourth, succinate dehydrogenase, uses FAD.

Question 3

NADH per glucose

Show solution

One glucose → 2 pyruvate → 2 acetyl-CoA, so 2 turns. 2 × 3 NADH = 6 NADH.

Question 4

ATP equivalents

Show solution

One turn: 3 × 2.5 + 1 × 1.5 + 1 = 10 ATP equivalents. Four turns: 4 × 10 = 40.

Question 5A liver cell has a high NADH/NAD⁺ ratio and plenty of ATP. What happens to isocitrate dehydrogenase?

Show solution

High NADH and high ATP both say the cell has enough energy. Both slow isocitrate dehydrogenase, so isocitrate and citrate can accumulate if their supply continues. Extra citrate can then leave the mitochondrion and slow PFK-1 in glycolysis.

Question 6Which step of the cycle makes a high-energy phosphate directly, without the electron transport chain?

Show solution

Step 5, succinyl-CoA synthetase, is the only substrate-level phosphorylation in the cycle. It makes 1 GTP or ATP per turn, depending on the tissue form of the enzyme.

Question 7Fluoroacetate is converted in cells to fluorocitrate, which blocks aconitase. Assuming upstream supply continues, which substrate tends to build up immediately?

Show solution

Aconitase consumes citrate. When its activity falls while citrate synthesis continues, citrate tends to accumulate. Supply to later cycle steps decreases, but actual intermediate concentrations also depend on other pathways.

Question 8No reaction in the citric acid cycle uses O₂. Why does sustained oxidative cycling stop in human mitochondria when oxygen runs out?

Show solution

In human mitochondria, O₂ accepts electrons at the end of the respiratory chain. Without it, NADH and enzyme-bound FADH₂ cannot be continuously reoxidized, so the oxidative cycle cannot keep running. Cytosolic lactate fermentation does not replace this mitochondrial electron flow.

Question 9Severe thiamine (vitamin B₁) deficiency lowers the activity of two enzyme complexes that use thiamine pyrophosphate. Which two?

Show solution

Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase work the same way and share the same cofactors: thiamine pyrophosphate, lipoate, CoA, FAD, and NAD⁺. With less thiamine, oxidation of pyruvate and α-ketoglutarate is impaired.

Question 10Human cells pull intermediates out of the cycle to build other molecules. Which reaction refills oxaloacetate?

Show solution

Pyruvate carboxylase uses bicarbonate (HCO₃⁻) as the CO₂ source to carboxylate pyruvate, making oxaloacetate. It needs ATP and biotin. Acetyl-CoA activates it, which makes sense: lots of acetyl-CoA means the cycle needs more oxaloacetate to accept it.

Frequently asked questions

How much ATP does the Krebs cycle make?

One turn makes 1 GTP or ATP directly, plus 3 NADH and 1 FADH₂. Once the electron transport chain cashes those in, one turn is worth about 10 ATP using the current values of 2.5 per NADH and 1.5 per FADH₂, or 12 ATP using the older values of 3 and 2. One glucose runs two turns.

Is the citric acid cycle the same as the Krebs cycle and the TCA cycle?

Yes. All three names mean the same pathway. Krebs honors Hans Krebs, citric acid comes from the first product, and TCA stands for tricarboxylic acid, because citrate has three carboxyl groups.

Does the citric acid cycle need oxygen?

No cycle step uses O₂ directly. In human mitochondria, sustained oxidative cycling depends on the respiratory chain reoxidizing NADH and enzyme-bound FADH₂, with O₂ as the final electron acceptor. Without oxygen, this flow cannot continue. Some microbes have different pathways and electron acceptors.

What is the rate-limiting step of the citric acid cycle?

Isocitrate dehydrogenase is the usual textbook answer, but control is shared among several steps and depends on conditions. ATP and NADH slow it, and ADP and calcium speed it up. Citrate synthase and α-ketoglutarate dehydrogenase are the two other one-way control points, and textbooks differ a little on which regulators they list, so check your course notes.

Is the citric acid cycle on the MCAT?

Yes. The AAMC content outline lists the citric acid cycle under bioenergetics and metabolism. Focus on the inputs and outputs, the regulated enzymes, and how the cycle connects to glycolysis and the electron transport chain. For the depth of enzyme and intermediate detail expected on your own exam, check your course outline.

Sources

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