Metabolism — citric acid cycle (step 1)
Citrate synthase: Claisen-type condensation
How the Citrate synthase works, in 3 steps. Each step shows the structures and the curved arrows that move the electrons.
In short. A 3-step mechanism. Typical reagents: Acetyl-CoA, oxaloacetate, citrate synthase (His274, His320, Asp375). Conditions: Mitochondrial matrix, pH ≈ 7.8, 37 °C. Bonds formed: C–C (aldol); citrate; CoA–SH; bonds broken: α C–H; thioester C–S (hydrolysis). Stereochemistry: Enolate attacks the si face of oxaloacetate; pro-S citrate. Rate and selectivity: Hydrolysis of citryl-CoA (ΔG°′ ≈ −32 kJ/mol overall) makes the step irreversible.
At a glance
Step 1: Base deprotonates acetyl-CoA
Acetyl-CoA's alpha carbon, made acidic by the adjacent thioester carbonyl, is deprotonated by an active-site base to form an enolate — the identical thioester-enolate chemistry already covered in fatty acid biosynthesis's decarboxylative Claisen condensation, just run here without the decarboxylation.
Step 2: Aldol addition to oxaloacetate
This enolate attacks oxaloacetate's ketone carbonyl carbon in an ordinary aldol addition, forming citryl-CoA — a new six-carbon skeleton built from a four-carbon and a two-carbon precursor.
Step 3: Thioester hydrolysis
Hydrolysis of the resulting thioester (ordinary nucleophilic acyl substitution by water) releases citrate and free CoA-SH — this aldol-addition-then-hydrolysis sequence is the committed first step of the citric acid cycle, and the hydrolysis step's substantial energy release is what makes the whole condensation irreversible.
Abbreviations and terms
- H2
- Hydrogen gas. Reductant for catalytic hydrogenation.
- CoA
- Coenzyme A. Thiol carrier that forms high-energy thioesters (acetyl-CoA).
How it compares with related reactions
| Citrate synthase | Aconitase | Fumarase | |
|---|---|---|---|
| Reagents | Acetyl-CoA, oxaloacetate, citrate synthase (His274, His320, Asp375) | Citrate, [4Fe-4S] cluster (Fe₄ binds OH), enzyme base (Ser) and acid | Fumarate, H₂O, active-site base (Ser/His) and acid |
| Conditions | Mitochondrial matrix, pH ≈ 7.8, 37 °C | Mitochondrial matrix, pH ≈ 7.4, 37 °C | Mitochondrial matrix, pH ≈ 7.4 |
| Stereochemistry | Enolate attacks the si face of oxaloacetate; pro-S citrate | Anti elimination; the intermediate flips so water adds to the opposite carbon | Anti addition on one face gives L-malate (S) only |
| Rate / selectivity | Hydrolysis of citryl-CoA (ΔG°′ ≈ −32 kJ/mol overall) makes the step irreversible | Near-equilibrium; citrate:isocitrate ≈ 90:10 at equilibrium | Near diffusion-limited; reversible |
Test yourself
Try to answer each question before opening it.
Which reagents and conditions are typical for the Citrate synthase?
Acetyl-CoA, oxaloacetate, citrate synthase (His274, His320, Asp375). Conditions: Mitochondrial matrix, pH ≈ 7.8, 37 °C.
Which bonds are formed and which are broken?
Formed: C–C (aldol); citrate; CoA–SH. Broken: α C–H; thioester C–S (hydrolysis).
What is the stereochemical outcome?
Enolate attacks the si face of oxaloacetate; pro-S citrate
What controls the rate or the selectivity?
Hydrolysis of citryl-CoA (ΔG°′ ≈ −32 kJ/mol overall) makes the step irreversible
Study guides that help with this reaction
- Carbonyl chemistry: addition, acyl substitution and enolates
- How to learn reaction mechanisms (without memorising everything)
- Curved-arrow notation: rules and the mistakes students make