Metabolism — citric acid cycle (step 7)
Fumarase: hydration of fumarate to malate
How the Fumarase 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: Fumarate, H₂O, active-site base (Ser/His) and acid. Conditions: Mitochondrial matrix, pH ≈ 7.4. Bonds formed: C–O (C–OH); C–H; bonds broken: C=C π; O–H (water). Stereochemistry: Anti addition on one face gives L-malate (S) only. Rate and selectivity: Near diffusion-limited; reversible.
At a glance
Step 1: Base activates water
An active-site base deprotonates a water molecule, generating a hydroxide nucleophile positioned right at fumarate's electron-poor alkene carbon — the alkene is activated toward addition by the adjacent electron-withdrawing carboxylate, much like a Michael acceptor.
Step 2: Conjugate addition, carbanion forms
That hydroxide attacks the alkene in a conjugate-addition-like step, generating a stabilized carbanion (enolate) intermediate at the other carbon.
Step 3: Protonation gives L-malate
Protonation of that carbanion gives malate — this hydration is stereospecific, adding water only across one specific face to give a single enantiomer, in sharp contrast to the non-stereospecific acid-catalyzed alkene hydration covered earlier in this library.
Abbreviations and terms
- L
- Ligand. A Lewis-base group (often a phosphine) bound to a metal centre.
- Ser
- Serine. Amino acid with a nucleophilic –OH.
- His
- Histidine. Amino acid whose imidazole acts as acid/base catalyst.
How it compares with related reactions
| Fumarase | Aconitase | Citrate synthase | |
|---|---|---|---|
| Reagents | Fumarate, H₂O, active-site base (Ser/His) and acid | Citrate, [4Fe-4S] cluster (Fe₄ binds OH), enzyme base (Ser) and acid | Acetyl-CoA, oxaloacetate, citrate synthase (His274, His320, Asp375) |
| Conditions | Mitochondrial matrix, pH ≈ 7.4 | Mitochondrial matrix, pH ≈ 7.4, 37 °C | Mitochondrial matrix, pH ≈ 7.8, 37 °C |
| Stereochemistry | Anti addition on one face gives L-malate (S) only | Anti elimination; the intermediate flips so water adds to the opposite carbon | Enolate attacks the si face of oxaloacetate; pro-S citrate |
| Rate / selectivity | Near diffusion-limited; reversible | Near-equilibrium; citrate:isocitrate ≈ 90:10 at equilibrium | Hydrolysis of citryl-CoA (ΔG°′ ≈ −32 kJ/mol overall) makes the step irreversible |
Test yourself
Try to answer each question before opening it.
Which reagents and conditions are typical for the Fumarase?
Fumarate, H₂O, active-site base (Ser/His) and acid. Conditions: Mitochondrial matrix, pH ≈ 7.4.
Which bonds are formed and which are broken?
Formed: C–O (C–OH); C–H. Broken: C=C π; O–H (water).
What is the stereochemical outcome?
Anti addition on one face gives L-malate (S) only
What controls the rate or the selectivity?
Near diffusion-limited; reversible
Study guides that help with this reaction
- How to learn reaction mechanisms (without memorising everything)
- Curved-arrow notation: rules and the mistakes students make