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 k(cat)/K(m) of 4.5 x 105 M-1 s- 1 under conditions of steady-state catalysis. Smaller or larger stem-loop RNAs have lower k(cat) values, but all have K(m) values of ~5 μM. Both the 10- and 18-base substrates have k(cat)/K(m) 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 k(cat)/K(m) 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 (K(i) = 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 pK(a) of the adenine analogue at N7. Binding of this analogue (K(i) = 9.4 μM) is weaker than substrate which indicates that the altered pK(a) 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 (K(i) = 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.
|Original language||English (US)|
|Number of pages||9|
|State||Published - Aug 18 1998|
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