Mapping the transition state for ATP hydrolysis: implications for enzymatic catalysis Original Research Article

these reactions are analogous to the phosphorylation of sugars and other biological alcohols and to the hydrolysis of ATP. The Brønsted βnucleophile value of 0.07 is small, indicating that there is little bond formation between the incoming nucleophile and the electrophilic phosphoryl group in the transition state. Coordination of Mg2+ has no measurable effect on this value. The Brønsted βleaving group group value of −1.1 for phosphoryl transfer to water from a series of phosphoanhydrides is large and negative, suggesting that the bond between phosphorous and the leaving group oxygen is largely broken in the transition state. Conclusions: Uncatalyzed hydrolysis of ATP in solution occurs via a dissociative, inetaphosphate-like transition state, with little bond formation between nucleophile and ATP and substantial cleavage of the bond between the γ-phosphoryl moiety and the ADP leaving group. Bound Mg2+ does not perturb the dissociative nature of the transition state, contrary to proposals that enzyme-bound metal ions alter this structure. The simplest expectation for phosphoryl transfer at the active site of enzymes thus entails a dissociative transition state. These results provide a basis for analyzing catalytic mechanisms for phosphoryl transfer.