O-18 Isotope Effects Support a ConcertedMechanism for Ribonuclease A

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Journal of the American Chemical Society

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Ribonuclease A catalyzes the cleavage of RNA in a two-step process. The phosphodiester bond is cleaved to yield a RNA strand with a free 5′ OH and another RNA strand with a 2′, 3′ cyclic monophosphate at the 3′ end. This cyclic intermediate is then hydrolyzed with enzymatic catalysis.1 Classically, the first step of cleavage of RNA by ribonuclease A has been reported to proceed via a concerted mechanism in which His-12 acts to deprotonate the nucleophilic 2′ OH and His-119 acts to protonate the leaving group.2 Chemical modification studies,3 pH-rate studies,4 and site directed mutagenesis studies of His-12 and His-1195 are consistent with general acid-base catalysis. However, the mechanism has become the subject of much debate as a result of the proposal of a phosphorane intermediate in the catalytic mechanism. This concept was based on data obtained in model studies utilizing cyclodextrin-bis(imidazole) compounds6 or imidazole or morpholine buffers as catalysts.7 We report here a direct study of the enzymatic reaction catalyzed by ribonuclease A to test for the presence of a phosphorane intermediate.

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