Ricin A-Chain: Kinetics, Mechanism, and RNA Stem−Loop Inhibitors

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Abstract
Ricin A-chain (RTA) catalyzes the depurination of a single adenine at position 4324 of 28S rRNA in a N-ribohydrolase reaction. The mechanism and specificity for RTA are examined using RNA stem−loop structures of 10−18 nucleotides which contain the required substrate motif, a GAGA tetraloop. At the optimal pH near 4.0, the preferred substrate is a 14-base stem−loop RNA which is hydrolyzed at 219 min-1 with a kcat/Km of 4.5 × 105 M-1 s-1 under conditions of steady-state catalysis. Smaller or larger stem−loop RNAs have lower kcat values, but all have Km values of ∼5 μM. Both the 10- and 18-base substrates have kcat/Km near 104 M-1 s-1. Covalent cross-linking of the stem has a small effect on the kinetic parameters. Stem−loop DNA (10 bases) of the same sequence is also a substrate with a kcat/Km of 0.1 that for RNA. Chemical mechanisms for enzymatic RNA depurination reactions include leaving group activation, stabilization of a ribooxocarbenium transition state, a covalent enzyme−ribosyl intermediate, and ionization of the 2‘-hydroxyl. A stem−loop RNA with p-nitrophenyl O-riboside at the depurination site is not a substrate, but binds tightly to the enzyme (Ki = 0.34 μM), consistent with a catalytic mechanism of leaving group activation. The substrate activity of stem−loop DNA eliminates ionization of the 2‘-hydroxyl as a mechanism. Incorporation of the C-riboside formycin A at the depurination site provides an increased pKa of the adenine analogue at N7. Binding of this analogue (Ki = 9.4 μM) is weaker than substrate which indicates that the altered pKa at this position is not an important feature of transition state recognition. Stem−loop RNA with phenyliminoribitol at the depurination site increases the affinity substantially (Ki = 0.18 μM). The results are consistent with catalysis occurring by leaving group protonation at ring position(s) other than N7 leading to a ribooxocarbenium ion transition state. Small stem−loop RNAs have been identified with substrate activity within an order of magnitude of that reported for intact ribosomes.