Enzyme catalysis — two-metal-ion mechanism
DNA polymerase: two-metal-ion nucleotidyl transfer
How the DNA polymerase 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: Primer–3′-OH, dNTP, 2 Mg²⁺, DNA polymerase (Asp carboxylates). Conditions: Aqueous, pH ≈ 7.5, 37 °C. Bonds formed: 3′-O–Pα (phosphodiester); PPi; bonds broken: Pα–O–Pβ anhydride; 3′-O–H. Stereochemistry: In-line attack, inversion at Pα. Rate and selectivity: kpol ≈ 10–300 s⁻¹ (chemistry step partly rate-limiting).
At a glance
Step 1: Metal A activates the 3′-OH
Two Mg²⁺ ions in the active site each coordinate part of the reacting groups: metal A lowers the pKa of the DNA primer's 3′-OH, helping deprotonate it without a separate enzymatic base.
Step 2: In-line attack on Pα; metal B on PPi
The now-activated 3′-alkoxide attacks the incoming nucleotide's α-phosphate in-line, forming the same pentacoordinate phosphorane transition state seen in any phosphoryl transfer, while metal B stabilizes the developing negative charge on the leaving pyrophosphate.
Step 3: Two metals stabilise the TS
Both metals also stabilize the pentacoordinate transition state electrostatically, dramatically accelerating the reaction — this two-metal-ion strategy is so effective it's shared by essentially every polymerase and nuclease in biology.
Abbreviations and terms
- B:
- Base. A proton acceptor.
- TS
- Transition state. The highest-energy point on the pathway between two minima; it cannot be isolated.
- pKa
- Acid-dissociation constant (–log). Lower pKa = stronger acid; used to judge which proton a base removes.
- DNA
- Deoxyribonucleic acid. The genetic polymer.
- dNTP
- Deoxynucleoside triphosphate. DNA building block.
- Asp
- Aspartate. Acidic amino acid.
How it compares with related reactions
| DNA polymerase | Aldolase | RNase A | |
|---|---|---|---|
| Reagents | Primer–3′-OH, dNTP, 2 Mg²⁺, DNA polymerase (Asp carboxylates) | Dihydroxyacetone phosphate (DHAP), glyceraldehyde 3-phosphate (G3P), Class I aldolase (Lys229) | RNA substrate, RNase A (His12 base, His119 acid, Lys41) |
| Conditions | Aqueous, pH ≈ 7.5, 37 °C | Aqueous, pH ≈ 7.5 | Aqueous, pH ≈ 6, 37 °C |
| Stereochemistry | In-line attack, inversion at Pα | Enzyme controls the face: gives the D-threo (3S,4R) product | In-line attack, inversion at P |
| Rate / selectivity | kpol ≈ 10–300 s⁻¹ (chemistry step partly rate-limiting) | Enamine formation/C–C bond formation rate-limiting | kcat/KM ≈ 10⁷ M⁻¹ s⁻¹; about 10¹¹-fold over uncatalysed |
Test yourself
Try to answer each question before opening it.
Which reagents and conditions are typical for the DNA polymerase?
Primer–3′-OH, dNTP, 2 Mg²⁺, DNA polymerase (Asp carboxylates). Conditions: Aqueous, pH ≈ 7.5, 37 °C.
Which bonds are formed and which are broken?
Formed: 3′-O–Pα (phosphodiester); PPi. Broken: Pα–O–Pβ anhydride; 3′-O–H.
What is the stereochemical outcome?
In-line attack, inversion at Pα
What controls the rate or the selectivity?
kpol ≈ 10–300 s⁻¹ (chemistry step partly rate-limiting)
Study guides that help with this reaction
- How to learn reaction mechanisms (without memorising everything)
- Curved-arrow notation: rules and the mistakes students make