Nucleophilic activation by positioning in phosphoryl transfer catalyzed bynucleoside diphosphate kinase

The nonenzymatic reaction of ATP with a nucleophile to generate ADP and a p hosphorylated product proceeds via a dissociative transition state with lit tle bond formation to the nucleophile. Consideration of the dissociative na ture of the nonenzymatic transition state leads to the following question: To what extent can the nucleophile be activated in enzymatic phosphoryl tra nsfer? We have addressed this question for the NDP kinase reaction. A mutan t form of the enzyme lacking the nucleophilic histidine (H122G) can be chem ically rescued for ATP attack by imidazole or other exogenous small nucleop hiles. The ATP reaction is 50-fold faster with the wild-type enzyme. which has an imidazole nucleophile positioned for reaction by a covalent bond, th an with H122G, which employs a noncovalently bound imidazole nucleophile [( k(cat)/K-M)(ATP)]. Further, a 4-fold advantage for imidazole positioned in the nucleophile binding pocket created by the mutation is suggested from co mparison of the reaction of H122G and ATP with an imidazole versus a water nucleophile, after correction for the intrinsic reactivities of imidazole a nd water toward ATP in solution. X-ray structural analysis shows no detecta ble rearrangement of the residues surrounding His 122 upon mutation to Gly 122. The overall rate effect of similar to 10(2)-fold for the covalent imid azole nucleophile relative to water is therefore attributed to positioning of the nucleophile with respect to the reactive phosphoryl group. This is u nderscored by the more deleterious effect of replacing ATP with ATP gamma S in the wild-type reaction than in the imidazole-rescued mutant reaction, a s follows. For the wild-type, ATP gamma S presumably disrupts positioning b etween nucleophile and substrate, resulting in a large thio effect of 300-f old, whereas precise alignment is already disrupted in the mutant because t here is no covalent bond to the nucleophile, resulting in a smaller thio ef fect of 10-fold. In summary, the results suggest a catalytic role for activ ation of the nucleophile by positioning in phosphoryl transfer catalyzed by NDP kinase.