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Enzyme catalysis — phosphodiester cleavage

RNase A: general acid/general base catalysis

How the RNase A works, in 3 steps. Each step shows the structures and the curved arrows that move the electrons.

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In short. A 3-step mechanism. Typical reagents: RNA substrate, RNase A (His12 base, His119 acid, Lys41). Conditions: Aqueous, pH ≈ 6, 37 °C. Bonds formed: 2′,3′-cyclic phosphate; 5′-OH; bonds broken: P–O5′ (RNA backbone); 2′-O–H. Stereochemistry: In-line attack, inversion at P. Rate and selectivity: kcat/KM ≈ 10⁷ M⁻¹ s⁻¹; about 10¹¹-fold over uncatalysed.

At a glance

ReagentsRNA substrate, RNase A (His12 base, His119 acid, Lys41)
Solvent / conditionsAqueous, pH ≈ 6, 37 °C
Bonds formed2′,3′-cyclic phosphate; 5′-OH
Bonds brokenP–O5′ (RNA backbone); 2′-O–H
StereochemistryIn-line attack, inversion at P
Rate / selectivitykcat/KM ≈ 10⁷ M⁻¹ s⁻¹; about 10¹¹-fold over uncatalysed

Step 1: His12 deprotonates 2′-OH

+general base+His12NHNBOOHOPOOOR′OHHis12NHNHBOOHOPOOOR′O1 · HIS12 DEPROTONATES 2′-OHHis12 (general base)BASERNA 2′-OHSUBSTRATEHis12-H⁺CATALYST2′-alkoxideNUCLEOPHILEThe 2′-O becomes alkoxide-like and aligns in-line with the P–O5′ bond.

One active-site histidine, acting as a general base, deprotonates RNA's 2′-OH, positioning it to attack the adjacent phosphate directly in-line with the 5′ leaving group.

Step 2: Attack on P; His119 protonates O5′

+attack,proton transferBOOHOPOOOR′OHNNHHis119BOOHOPOOOR′O2 · ATTACK ON P; HIS119 PROTONATES O5′2′-alkoxide + phosphateNUCLEOPHILEHis119-H⁺ (general acid)ACIDpentacoordinate phosphoraneINTERMEDIATEMetal-free: His12 and His119 do the acid/base work; Lys41 stabilises the phosphorane.

That activated 2′-oxygen attacks phosphorus, forming a pentacoordinate phosphorane exactly as in any phosphoryl transfer, while a second histidine acts as a general acid, protonating the departing 5′-oxygen to help it leave.

Step 3: Collapse to the cyclic phosphate

collapse+BOOHOPOOOR′OBOOHOPOOOHOR′3 · COLLAPSE TO THE CYCLIC PHOSPHATEphosphoraneINTERMEDIATE2′,3′-cyclic phosphatePRODUCT5′-OH RNA fragmentLEAVING GROUPWater and His12 then hydrolyse the cyclic phosphate to 3′-phosphate (turnover).

The phosphorane collapses to a 2′,3′-cyclic phosphate intermediate, cleaving the RNA backbone — two histidines working as an acid/base pair is a textbook case of enzymatic catalysis accelerating exactly the same phosphorus chemistry covered in the step before.

Abbreviations and terms

R
R group. Any carbon substituent (alkyl, aryl…) whose identity does not matter for the mechanism.
M
Metal. A generic metal centre.
RNA
Ribonucleic acid. Single-stranded nucleic acid.
RNase
Ribonuclease. Enzyme that cleaves RNA.

How it compares with related reactions

RNase AAldolaseDNA polymerase
ReagentsRNA substrate, RNase A (His12 base, His119 acid, Lys41)Dihydroxyacetone phosphate (DHAP), glyceraldehyde 3-phosphate (G3P), Class I aldolase (Lys229)Primer–3′-OH, dNTP, 2 Mg²⁺, DNA polymerase (Asp carboxylates)
ConditionsAqueous, pH ≈ 6, 37 °CAqueous, pH ≈ 7.5Aqueous, pH ≈ 7.5, 37 °C
StereochemistryIn-line attack, inversion at PEnzyme controls the face: gives the D-threo (3S,4R) productIn-line attack, inversion at Pα
Rate / selectivitykcat/KM ≈ 10⁷ M⁻¹ s⁻¹; about 10¹¹-fold over uncatalysedEnamine formation/C–C bond formation rate-limitingkpol ≈ 10–300 s⁻¹ (chemistry step partly rate-limiting)

Test yourself

Try to answer each question before opening it.

Which reagents and conditions are typical for the RNase A?

RNA substrate, RNase A (His12 base, His119 acid, Lys41). Conditions: Aqueous, pH ≈ 6, 37 °C.

Which bonds are formed and which are broken?

Formed: 2′,3′-cyclic phosphate; 5′-OH. Broken: P–O5′ (RNA backbone); 2′-O–H.

What is the stereochemical outcome?

In-line attack, inversion at P

What controls the rate or the selectivity?

kcat/KM ≈ 10⁷ M⁻¹ s⁻¹; about 10¹¹-fold over uncatalysed

Study guides that help with this reaction

Related mechanisms

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