RICIN A-CHAIN - KINETICS, MECHANISM, AND RNA STEM-LOOP INHIBITORS

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 o f 10-18 nucleotides which contain the required substrate motif, a GAGA tetraloop. At the optimal pH near 4.0, the preferred substrate is a 1 4-base stem-loop RNA which is hydrolyzed at 219 min(-1) with a k(cat)/ K-m of 4.5 x 10(5) M-1 s(-1) under conditions of steady-state catalysi s. Smaller or larger stem-loop RNAs have lower k(cat) values, but all have K-m values of similar to 5 mu M. Both the 10- and 18-base substra tes have k(cat)/K-m near 10(4) M-1 s(-1). Covalent cross-linking of th e 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 r eactions include leaving group activation, stabilization of a ribooxoc arbenium transition state, a covalent enzyme-ribosyl intermediate, and ionization of the 2'-hydroxyl. A stem-loop RNA with p-nitrophenyl O-r iboside at the depurination site is not a substrate, but binds tightly to the enzyme (K-i = 0.34 mu M), consistent with a catalytic mechanis m of leaving group activation. The substrate activity of stem-loop DNA eliminates ionization of the 2'-hydroxyl as a mechanism. Incorporatio n of the C-riboside formycin A at the depurination site provides an in creased pK(a) of the adenine analogue at N7. Binding of this analogue (K-i = 9.4 mu M) is weaker than substrate which indicates that the alt ered pK(a) at this position is not an important feature of transition state recognition. Stem-loop RNA with phenyliminoribitol at the depuri nation site increases the affinity substantially (K-i = 0.18 mu M). Th e results are consistent with catalysis occurring by leaving group pro tonation at ring position(s) other than N7 leading to a ribooxocarbeni um ion transition state. Small stem-loop RNAs have been identified wit h substrate activity within an order of magnitude of that reported for intact ribosomes.