Kinetic isotope effects and stereochemical studies on a ribonuclease model: Hydrolysis reactions of uridine 3 '-nitrophenyl phosphate

The reactions of a ribonuclease model substrate, the compound uridine-3'-p- nitrophenyl phosphate, have been examined using heavy-atom isotope effects and stereochemical analysis. The cyclization of this compound is subject to catalysis by general base (by imidazole buffer), specific base (by carbona te buffer), and by acid. All three reactions proceed by the same mechanisti c sequence, via cyclization to cUMP, which is stable under basic conditions but which is rapidly hydrolyzed to a mixture of 2'- and 3'-UMP under acid conditions. The isotope effects indicate that the specific base-catalyzed r eaction exhibits an earlier transition state with respect to bond cleavage to the leaving group compared to the general base-catalyzed reaction. Stere ochemical analysis indicates that both of the base-catalyzed reactions proc eed with the same stereochemical outcome. It is concluded that the differen ce in the nucleophile in the two base-catalyzed reactions results in a diff erence in the transition state structure but both reactions are most likely concerted, with no phosphorane intermediate. The N-15 isotope effects were also measured for the reaction of the substrate with ribonuclease A. The r esults indicate that considerably less negative charge develops on the leav ing group in the transition state than for the general base-catalyzed react ion in solution. (C) 2000 Academic Press.