Kinetics and mechanism in RNA cleavage.

Abstract

Experimental studies on the cleavage of various RNA molecules--poly(U), 3',5''-UpU, 2',5''-UpU, and 3',5''-ApA--catalyzed by imidazole buffers show that there is a sequential bifunctional mechanism. One catalyst species converts the substrate to an intermediate phosphorane, and the second catalyst converts the phosphorane to cleavage products. Detailed steady-state kinetics are presented to determine all mechanisms that are consistent with the findings. Choice among these possibilities can be made considering other experimental evidence about the catalysis of an isomerization reaction that accompanies cleavage. It is concluded that all acceptable mechanisms involve action of the second catalyst on a phosphorane monoanion; it can be formed directly from the substrate in the first step or by proton equilibrations involving either the substrate or the phosphorane intermediate. The relationship of these conclusions to the likely mechanism of action of the enzyme ribonuclease is briefly discussed.