What is Dr. Zubair Khalid's research focus?

Dr. Zubair Khalid specializes in molecular virology, mRNA vaccine development, and computational biology, with a focus on avian pathogens like IBDV and Avian Reovirus.

Where is Dr. Zubair Khalid currently working?

Dr. Zubair Khalid is a Postdoctoral Research Associate at the University of Maryland (UMD), specifically within the Department of Animal and Avian Sciences.

Citric Acid Cycle: Steps and Diagram Explained

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-09-20

The citric acid cycle is an eight-step mitochondrial pathway that oxidizes the two-carbon acetyl group of acetyl-CoA to two molecules of carbon dioxide while reducing NAD+ and FAD to NADH and FADH2. It is also called the tricarboxylic acid cycle, the TCA cycle, or the Krebs cycle, and it is the final common oxidation route for carbohydrates, fats, and most amino acids.

The cycle matters because it is the metabolic hub of the cell. Every carbon that enters from glucose, fatty acids, or amino acid breakdown is funneled through acetyl-CoA and then through these eight reactions. The reduced cofactors it produces carry electrons to the respiratory chain, and several of its intermediates double as biosynthetic precursors and as signaling molecules that shape immune and inflammatory responses [1]. A working knowledge of the citric and cycle reactions is therefore foundational for biochemistry, microbiology, cell biology, and metabolic engineering.

Why This Pathway Sits at the Center of Metabolism

Three features explain the central position of the citric acid cycle.

First, it is amphibolic. That term means the pathway serves both catabolic and anabolic roles. It breaks down acetyl-CoA for energy, and it also supplies precursors for biosynthesis. Oxaloacetate feeds gluconeogenesis, alpha-ketoglutarate feeds amino acid synthesis, and succinyl-CoA feeds heme synthesis.

Second, it is a convergence point. Glycolysis produces pyruvate, pyruvate dehydrogenase converts pyruvate to acetyl-CoA, and fatty acid beta-oxidation also produces acetyl-CoA. Amino acid catabolism feeds in at several points. Computational models that integrate glycolysis, the pentose phosphate pathway, fatty acid beta-oxidation, and the tricarboxylic cycle into one network show how tightly these routes are coupled [2].

Third, its intermediates are bioactive. Succinate, fumarate, itaconate, malate, and oxaloacetate accumulate during inflammation and act as metabolic danger signals that regulate immunity through receptor-dependent and receptor-independent mechanisms [1]. Succinate and alpha-ketoglutarate exert opposing signaling effects on macrophage phenotype, with succinate promoting inflammation and alpha-ketoglutarate favoring an anti-inflammatory state [3].

Quick Comparison: Names and Related Terms

Term	What it refers to	Notes
Citric acid cycle	The canonical name	Named for citrate, the first intermediate
TCA cycle	Tricarboxylic acid cycle	Refers to the three carboxyl groups on citrate
Krebs cycle	Honorific name	Named after Hans Krebs
Citrate in Krebs cycle	The specific first intermediate	Citrate is the entry point for acetyl-CoA carbon
Glyoxylate shunt	Bypass of two decarboxylation steps	Used by plants and bacteria, not by mammals
Reductive TCA cycle	Reverse carbon-fixing route	Found in some autotrophic bacteria [4]
2-methylcitrate cycle	Propionate oxidation route	A separate pathway that resembles the TCA cycle [5]

The Eight Steps in Order

Each turn of the cycle consumes one acetyl-CoA and regenerates oxaloacetate, so the cycle is catalytic. The eight reactions are numbered below in the order they occur.

Step 1: Citrate Synthase

Citrate synthase condenses the two-carbon acetyl group of acetyl-CoA with the four-carbon oxaloacetate to form the six-carbon citrate. This is an irreversible, committed step and a major control point. The reaction is a Claisen condensation, and it releases free CoA. Because oxaloacetate is regenerated at the end of the cycle, it acts catalytically, and its concentration is normally kept low.

Step 2: Aconitase

Aconitase isomerizes citrate to isocitrate through a dehydration step that forms cis-aconitate, followed by rehydration. The enzyme contains an iron-sulfur cluster that positions the substrate and abstracts a proton. The net effect is the movement of a hydroxyl group from one carbon to an adjacent carbon, which sets up the oxidative decarboxylations that follow. Aconitase is the target of industrial strategies to redirect carbon flux, and partial inhibition of the enzyme in Aspergillus niger increased citric acid accumulation from about 26 g/L to nearly 49 g/L in one study [6].

Step 3: Isocitrate Dehydrogenase

Isocitrate dehydrogenase performs the first oxidative decarboxylation. It converts isocitrate to alpha-ketoglutarate, releases the first molecule of carbon dioxide, and reduces NAD+ to NADH. This is a rate-limiting step and a key regulated point. In many organisms there are both NAD+-dependent and NADP+-dependent isoforms, and their relative activity shapes whether carbon continues through the cycle or is diverted to lipid synthesis [7]. The IDH3B subunit of the enzyme has been shown to be upregulated by viral proteins that push cells toward higher glutaminolysis and TCA cycle flux [8].

Step 4: Alpha-Ketoglutarate Dehydrogenase

The alpha-ketoglutarate dehydrogenase complex performs the second oxidative decarboxylation. It converts alpha-ketoglutarate to succinyl-CoA, releases the second molecule of carbon dioxide, and reduces NAD+ to NADH. This is a large multienzyme complex that resembles pyruvate dehydrogenase and requires thiamine pyrophosphate, lipoic acid, CoA, FAD, and NAD+. This enzyme is a recognized rate-limiting step in the cycle and a potent source of mitochondrial hydrogen peroxide, which allows it to influence signaling as well as flux [3]. Its product, succinyl-CoA, is used for protein succinylation reactions that affect gene expression programs in cancer cells [9].

Step 5: Succinyl-CoA Synthetase

Succinyl-CoA synthetase (also called succinate thiokinase) converts succinyl-CoA to succinate. The energy released by breaking the high-energy thioester bond drives substrate-level phosphorylation of GDP to GTP (in mammals) or of ADP to ATP (in some bacteria and plants). This is the only step in the cycle that directly generates a high-energy phosphate bond. The GTP produced can be converted to ATP by nucleoside diphosphate kinase.

Step 6: Succinate Dehydrogenase

Succinate dehydrogenase oxidizes succinate to fumarate and reduces FAD to FADH2. This enzyme is unusual because it is embedded in the inner mitochondrial membrane and is also part of the respiratory chain as Complex II. It is the only TCA cycle enzyme that is membrane-bound. Because it uses FAD rather than NAD+ as its electron acceptor, it feeds electrons into the ubiquinone pool directly. Succinate accumulation has signaling consequences, since succinate promotes inflammatory macrophage phenotypes and activates the receptor GPR91 [3].

Step 7: Fumarase

Fumarase (fumarate hydratase) catalyzes the reversible hydration of fumarate to malate. The enzyme adds water across the double bond of fumarate in a stereospecific manner to produce L-malate. Fumarate itself has emerged as an immunometabolite that can act as a metabolic danger signal when it accumulates [1].

Step 8: Malate Dehydrogenase

Malate dehydrogenase oxidizes malate to oxaloacetate and reduces NAD+ to NADH. This is the final step, and it regenerates the oxaloacetate needed for the next turn of the cycle. The reaction is strongly endergonic as written, but it is pulled forward because citrate synthase consumes oxaloacetate rapidly, keeping its steady-state concentration very low. Malate dehydrogenase activity has been used as a marker of central carbon metabolism in clinical samples, and elevated activity was detected in gingivitis tissue in one proteomic and enzymatic study [10]. In industrial fermentation work, temporal shifts in malate dehydrogenase and isocitrate dehydrogenase activity track the redirection of carbon toward lipid synthesis [11].

The Cycle as a Flow

The diagram below shows the order of the eight reactions and the points where carbon leaves as carbon dioxide and where reducing equivalents are captured.

flowchart TD
    A[Acetyl-CoA plus Oxaloacetate] --> B[Citrate]
    B --> C[Isocitrate]
    C --> D[Alpha-ketoglutarate]
    D --> E[Succinyl-CoA]
    E --> F[Succinate]
    F --> G[Fumarate]
    G --> H[Malate]
    H --> I[Oxaloacetate regenerated]
    C --> J[NADH and CO2 released]
    D --> K[NADH and CO2 released]
    F --> L[FADH2 captured]
    H --> M[NADH captured]

Numbered Reference Table

The table below lists each step with its enzyme, substrate, product, and the cofactor or coenzyme involved.

Step	Enzyme	Substrate	Product	Cofactor or coenzyme
1	Citrate synthase	Acetyl-CoA + oxaloacetate	Citrate + CoA	None (condensation)
2	Aconitase	Citrate	Isocitrate	Iron-sulfur cluster
3	Isocitrate dehydrogenase	Isocitrate	Alpha-ketoglutarate + CO2	NAD+ to NADH
4	Alpha-ketoglutarate dehydrogenase	Alpha-ketoglutarate	Succinyl-CoA + CO2	NAD+, CoA, TPP, lipoate, FAD
5	Succinyl-CoA synthetase	Succinyl-CoA	Succinate	GDP to GTP (or ADP to ATP)
6	Succinate dehydrogenase	Succinate	Fumarate	FAD to FADH2
7	Fumarase	Fumarate	Malate	None (hydration)
8	Malate dehydrogenase	Malate	Oxaloacetate	NAD+ to NADH

Yield Per Turn and ATP Equivalents

One turn of the cycle per acetyl-CoA yields 3 NADH, 1 FADH2, 1 GTP, and 2 CO2. The two carbon dioxide molecules come from the two oxidative decarboxylation steps, catalyzed by isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase.

The ATP equivalents depend on how the reducing equivalents are handled. Each NADH yields roughly 2.5 ATP and each FADH2 yields roughly 1.5 ATP when electrons pass through the respiratory chain, and the GTP is equivalent to 1 ATP. That gives approximately 10 ATP per acetyl-CoA. The exact number depends on the shuttle used to move reducing equivalents from the cytosol into the mitochondrion, because the glycerol-3-phosphate shuttle and the malate-aspartate shuttle deliver electrons at different points and therefore give different yields. The commonly quoted range is about 10 ATP per acetyl-CoA, and the number is approximate rather than fixed.

Regulation of the Cycle

Two steps are rate-limiting and heavily regulated: citrate synthase and isocitrate dehydrogenase. Alpha-ketoglutarate dehydrogenase is also a control point, and it is often grouped with these two in textbook discussions of flux control.

The main regulators are ATP, NADH, and calcium. High ATP and high NADH signal that the cell has plenty of energy and reducing power, so they inhibit the cycle. High ADP and high NAD+ signal energy demand and activate the cycle. Calcium activates key enzymes when muscle contraction or other energy-demanding processes raise cytosolic calcium. This makes the cycle responsive to the real-time energy state of the cell rather than running at a fixed rate.

The alpha-ketoglutarate dehydrogenase complex is a nexus for regulation in immune cells. Its activity controls the availability of alpha-ketoglutarate and succinate, and these two metabolites have opposite effects on macrophage polarization, with alpha-ketoglutarate favoring an anti-inflammatory state and succinate favoring a pro-inflammatory state [3].

Anaplerotic Reactions

The cycle consumes intermediates for biosynthesis, so it needs reactions that replenish them. These are called anaplerotic reactions, from the Greek for "filling up."

The most important anaplerotic enzyme in many organisms is pyruvate carboxylase, which converts pyruvate to oxaloacetate. Phosphoenolpyruvate carboxylase (Ppc) is another key anaplerotic enzyme, and it fixes bicarbonate onto phosphoenolpyruvate to form oxaloacetate. In Escherichia coli, the small RNA OmrA inhibits translation of Ppc, and this repression reduces TCA cycle flux enough to impair growth in glucose minimal medium. Supplementing the medium with glutamate, glutamine, or downstream TCA cycle metabolites restores growth, which confirms that the limitation is anaplerotic carbon [12].

In plants, phosphoenolpyruvate carboxylase variants with altered allosteric regulation release more malate from roots, and the same amino acid changes improve enzyme output when introduced into maize isozymes [13]. In metabolic engineering, anaplerotic flux is a common target for redirecting carbon toward products such as malate [14].

How the Cycle Is Measured in Practice

Several standard approaches are used to observe the cycle in the laboratory.

Enzyme activity assays measure the rate of individual reactions. These assays typically follow the appearance of NADH at 340 nm or the reduction of an artificial electron acceptor. Kinetic parameters such as Km and Vmax are determined by varying substrate concentration. For example, 2-methylcitrate synthase from Eimeria tenella was characterized with a Km of 5.239 mM for propionyl-CoA, 1.102 micromolar for oxaloacetic acid, and 5.999 micromolar for acetyl-CoA, with optimal activity at 41 degrees Celsius and pH 8.0 [5].

Isotope tracing with 13C-labeled substrates follows carbon through the cycle. In E. coli engineered for malate production, 13C-labeled glucose tracing showed that malate-producing strains had very high flux through the glyoxylate shunt with almost no flux through the isocitrate dehydrogenase reaction [14]. In CHO-K1 cell culture, 13C-labeled disaccharides were used to track incorporation of carbon into TCA cycle intermediates, which helped identify the route by which maltose is metabolized [15].

Proteomic and metabolomic profiling measures enzyme abundance and metabolite levels together. In a study of gingivitis, proteomic analysis of oral lavage identified malate dehydrogenase and triosephosphate isomerase among the top hits, and enzymatic assays confirmed elevated activity of both enzymes in diseased samples [10]. In a study of the oleaginous thraustochytrid Aurantiochytrium, dynamic metabolic modeling combined with enzyme activity analysis revealed temporal shifts in isocitrate dehydrogenase and malate dehydrogenase that accompanied increased lipid synthesis in a mutant strain [11].

Oxygen consumption measurements with instruments such as the NextGen-O2k allow real-time tracking of mitochondrial respiration while simultaneously monitoring membrane potential, NADH, and the ubiquinone redox state. This approach was used to show that proline oxidation can sustain ATP production when Complex I is inhibited, because proline dehydrogenase transfers electrons directly to ubiquinone [16].

Comparative and Applied Relevance

The citric acid cycle is not identical in all organisms, and the differences matter for both basic science and industry.

In bacteria, the cycle supports both energy production and biosynthesis, and its flux is tuned by small RNAs and by the availability of anaplerotic carbon [12]. In the thermophilic autotrophic bacterium Desulfurobacterium thermolithotrophum, a reductive TCA cycle operates in reverse to fix carbon dioxide, and hydrogen availability controls the abundance of the enzymes involved [4]. In the fungus Candida albicans, mitochondrial proteins that coordinate TCA cycle enzymes are required for utilization of N-acetylglucosamine and for the switch between commensal and pathogenic states [17].

In industrial biotechnology, the cycle is both a source of products and a competing pathway. Aspergillus niger is used for citric acid production, and blocking aconitase activity with potassium ferrocyanide and methanol increased citric acid output from about 26 g/L to nearly 49 g/L in a mutant strain [6]. In E. coli, deleting malic enzymes and dehydrogenases while expressing a malate-insensitive PEP carboxylase and an NADH-insensitive citrate synthase produced malate at a yield of 0.82 mol per mol of glucose [14]. In Yarrowia lipolytica, engineering an acetate uptake shortcut that feeds acetyl-CoA directly into polyketide synthesis produced triacetic acid lactone at titers up to 4.76 g/L, which shows how the cycle competes with product pathways for the same acetyl-CoA pool [18].

In immunology, TCA cycle metabolites are now recognized as signaling molecules. Succinate, fumarate, and itaconate accumulate during inflammation and act as metabolic danger signals, and the concept of metabolic DAMPs has been introduced to describe this class of molecules [1]. The alpha-ketoglutarate dehydrogenase complex sits at the center of this regulation because it controls the balance between alpha-ketoglutarate and succinate [3].

Common Mistakes and Limitations

Students frequently confuse the citric acid cycle with the electron transport chain. The cycle produces NADH and FADH2, and the respiratory chain oxidizes them. They are separate processes that are linked by the reduced cofactors.

Another common error is counting the ATP yield as exactly 10 per acetyl-CoA. The true value depends on the shuttle used to transfer cytosolic reducing equivalents into the mitochondrion, so the number is approximate.

A third mistake is assuming that the cycle always runs at maximum rate. It is tightly regulated by ATP, NADH, and calcium, and flux rises and falls with the energy state of the cell.

A fourth mistake is treating the cycle as a closed loop. It is not. Intermediates are constantly withdrawn for biosynthesis and must be replenished by anaplerotic reactions.

A fifth mistake is confusing the citric acid cycle with the glyoxylate shunt. The shunt bypasses the two decarboxylation steps and allows net carbon incorporation, and it operates in plants and bacteria but not in mammals.

Finally, the cycle does not operate in isolation. Its flux is coordinated with glycolysis, the pentose phosphate pathway, and fatty acid oxidation, and computational models that integrate these routes show how changes in one pathway propagate to the others [2]. Individual metabolic states vary, and any clinical interpretation of metabolic data requires professional evaluation.

Quick Review

The citric acid cycle has eight steps, and each turn consumes one acetyl-CoA and regenerates oxaloacetate.
The eight enzymes in order are citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase.
One turn yields 3 NADH, 1 FADH2, 1 GTP, and 2 CO2.
The ATP equivalent is about 10 per acetyl-CoA, and the exact value depends on the shuttle used.
Citrate synthase and isocitrate dehydrogenase are rate-limiting and are regulated by ATP, NADH, and calcium.
Succinate dehydrogenase is the only membrane-bound enzyme in the cycle and is also Complex II of the respiratory chain.
Anaplerotic reactions such as pyruvate carboxylase and PEP carboxylase replenish cycle intermediates.

Frequently Asked Questions

What is the citric acid cycle in simple terms?

The citric acid cycle is a series of eight enzyme-catalyzed reactions in the mitochondrial matrix that oxidize acetyl-CoA to carbon dioxide while producing NADH, FADH2, and GTP. It is the central pathway for energy extraction from carbohydrates, fats, and proteins.

Why is it called the TCA cycle?

TCA stands for tricarboxylic acid, which refers to the three carboxyl groups present on citrate, the first intermediate of the cycle. The names citric acid cycle, TCA cycle, and Krebs cycle all refer to the same pathway.

How many ATP does one turn of the citric acid cycle produce?

One turn produces approximately 10 ATP equivalents per acetyl-CoA. This comes from 3 NADH, 1 FADH2, and 1 GTP, with the exact yield depending on the shuttle used to transfer reducing equivalents into the mitochondrion.

Which enzymes are rate-limiting in the citric acid cycle?

Citrate synthase and isocitrate dehydrogenase are the primary rate-limiting enzymes. Alpha-ketoglutarate dehydrogenase is also a major control point. All three are regulated by ATP, NADH, and calcium.

What are anaplerotic reactions?

Anaplerotic reactions are reactions that replenish citric acid cycle intermediates that have been withdrawn for biosynthesis. Pyruvate carboxylase and phosphoenolpyruvate carboxylase are common examples.

Does the citric acid cycle occur in all organisms?

The oxidative citric acid cycle occurs in aerobic organisms, including mammals, plants, fungi, and many bacteria. Some anaerobic and autotrophic organisms use a reductive TCA cycle that runs in reverse to fix carbon dioxide.