Probing the mechanism of enzymatic phosphoryl transfer with a chemical trick

Nucleophilic substitutions at phosphorus comprise one of the most important classes of reactions in biology. Phosphate diester substitution reactions are catalyzed by nucleases and polymerases and are critical in DNA replication and transcription. Phosphate monoester (phosphoryl transfer) reactions are catalyzed by GTPases, ATPases, protein, and small molecule kinases, protein, and small molecule phosphatases. These enzymes play diverse roles in energy regulation, cell signaling, ion and small molecule transport, and nucleotide synthesis. There have been intensive efforts to try to understand the details of phosphoryl transfer reactions extending from nonenzymatic (or enzyme model) systems to the mechanisms of the enzymatic reactions, as exemplified by the study by Cho et al. in the current issue of PNAS (1). A full and convincing explanation at the quantum mechanical level has not been made as to why dissociative transition states should be preferred for nonenzymatic phosphoryl transfer reactions. From the many decades of work on small molecules, consensus for a dissociative transition state, akin to an SN1 reaction in organic chemistry, has been reached by most investigators (2, 3). In such a transition state, the bond between the phosphorus and leaving group has largely broken before the formation of a bond between the incoming nucleophile and phosphorus. The role of the nucleophile is diminished, and the formation of a highly reactive metaphosphate-like species is central in a dissociative transition state. This contrasts with the transition state of an associative mechanism, which occurs in phosphate triester substitution reactions, in which a pentavalent-like species is generated. In associative transition states, there is a significant degree of bond formation between the incoming nucleophile and the attacked phosphorus before leaving group departure. Although a full and convincing explanation at the quantum mechanical level has not yet been made as …