Metabolism — glycolysis (step 6)
GAPDH: oxidative phosphorylation of an aldehyde
How the GAPDH 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: Glyceraldehyde-3-phosphate (G3P), NAD⁺, inorganic phosphate (Pi), GAPDH (Cys149, His176). Conditions: Aqueous, pH ≈ 7.4, 37 °C (cytosol). Bonds formed: S–C thiohemiacetal; thioester; NADH; C–OPO₃ acyl phosphate; bonds broken: aldehyde C=O π; C–H (hydride); thioester C–S. Stereochemistry: Hydride delivered to one face of NAD⁺ (pro-R). Rate and selectivity: Fast and reversible; couples oxidation to phosphate capture.
At a glance
Step 1: Cys thiolate adds to the aldehyde
An active-site cysteine's thiol attacks the aldehyde carbon of glyceraldehyde-3-phosphate, forming a covalent thiohemiacetal — the sulfur analog of the hemiacetal chemistry seen in sugar ring-opening, just with a protein nucleophile.
Step 2: Hydride transfer to NAD⁺
A hydride transfers from that thiohemiacetal carbon directly to NAD⁺, oxidizing the intermediate to a high-energy thioester while reducing NAD⁺ to NADH — thioesters are substantially higher in energy than ordinary oxygen esters, which is exactly what this step is engineered to generate.
Step 3: Phosphate displaces the cysteine
Inorganic phosphate then attacks that thioester's carbonyl carbon (ordinary nucleophilic acyl substitution), displacing the cysteine thiolate and releasing 1,3-bisphosphoglycerate — this step is how glycolysis captures the energy of aldehyde oxidation directly as a high-energy phosphate bond, one step before it's transferred to ADP to make ATP.
Abbreviations and terms
- R
- R group. Any carbon substituent (alkyl, aryl…) whose identity does not matter for the mechanism.
- H2
- Hydrogen gas. Reductant for catalytic hydrogenation.
- ATP
- Adenosine triphosphate. The cell’s energy currency; transfers phosphate groups.
- ADP
- Adenosine diphosphate. ATP after losing one phosphate.
- NAD+
- Nicotinamide adenine dinucleotide (oxidised). Hydride-accepting coenzyme in oxidations.
- NADH
- Nicotinamide adenine dinucleotide (reduced). Hydride-donating coenzyme.
- GAPDH
- Glyceraldehyde-3-phosphate dehydrogenase. Glycolytic enzyme oxidising G3P with NAD⁺.
How it compares with related reactions
| GAPDH | Enolase | Hexokinase | |
|---|---|---|---|
| Reagents | Glyceraldehyde-3-phosphate (G3P), NAD⁺, inorganic phosphate (Pi), GAPDH (Cys149, His176) | 2-Phosphoglycerate, 2 Mg²⁺, enolase (Lys345 base, Glu211 acid) | Glucose, Mg·ATP, hexokinase |
| Conditions | Aqueous, pH ≈ 7.4, 37 °C (cytosol) | Cytosol, pH ≈ 7, 37 °C | Aqueous, pH ~7.4, 37 °C |
| Stereochemistry | Hydride delivered to one face of NAD⁺ (pro-R) | Anti elimination of H and OH; reversible | In-line SN2-like, inversion at P |
| Rate / selectivity | Fast and reversible; couples oxidation to phosphate capture | ΔG°′ ≈ +1.8 kJ/mol; PEP has a very high phosphoryl-transfer potential | Irreversible (ΔG°′ ≈ −17 kJ/mol); glucose induces domain closure |
Test yourself
Try to answer each question before opening it.
Which reagents and conditions are typical for the GAPDH?
Glyceraldehyde-3-phosphate (G3P), NAD⁺, inorganic phosphate (Pi), GAPDH (Cys149, His176). Conditions: Aqueous, pH ≈ 7.4, 37 °C (cytosol).
Which bonds are formed and which are broken?
Formed: S–C thiohemiacetal; thioester; NADH; C–OPO₃ acyl phosphate. Broken: aldehyde C=O π; C–H (hydride); thioester C–S.
What is the stereochemical outcome?
Hydride delivered to one face of NAD⁺ (pro-R)
What controls the rate or the selectivity?
Fast and reversible; couples oxidation to phosphate capture
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