Quantitative evidence for the mechanism of RNA cleavage by enzyme mimics. Cleavage and isomerization of UpU by morpholine buffers

Article abstract of DOI:10.1021/ja00076a031

The cleavage of 3′,5″-UpU to form the 2′,3′-cyclic phosphate and uridine and the simultaneous isomerization of the substrate to 2′,5″-UpU have been studied in morpholine buffer. The total rate of cleavage with morpholine buffer at 90/10 base to acid ratio and constant ionic strength shows an increase with increasing buffer concentration up to 1 M, but the isomerization rate shows a decrease, followed by a constant rate below that of the uncatalyzed process. A similar increase in rate of cleavage - but decrease in rate of isomerization - is also seen with a 95/5 morpholine/ morpholinium buffer and with data that can fit the theoretical equations using those parameters that are common for the 90/10 buffer. With 80/20 morpholine/morpholinium the negative effect of buffer on the rate of isomerization is smaller and not clearly seen. These observations are consistent with the predictions from a kinetic treatment of a previously proposed mechanism: the substrate is converted to a phosphorane intermediate by buffer acid in the common first steps of both processes, but the paths then branch from that intermediate. The cleavage path shows buffer base catalysis, but the isomerization path does not. At higher buffer concentrations an additional catalytic process seems to have been detected. The dependences of the buffer-catalyzed reactions on buffer ratios are also consistent with those reported earlier for imidazole catalysis. The early imidazole data fit the theoretical predictions of our mechanistic treatment. They are also consistent with our mechanism according to a criterion suggested by a critic. The results confirm previous findings. They also furnish detailed evidence for some of the steps that were previously only suggested.