Reversible phosphorylation of proteins is a post-translational modification that regulates diverse biological processes. The molecular mechanism underlying phosphoryl transfer catalyzed by enzymes remains a subject of active debate. In particular, the nature of transition state (TS), whether it has an associative or dissociative character, has been one of the most controversial issues. Structural evidence supports associative TS, whereas physical organic studies point to a dissociative character. Here we perform hybrid quantum mechanics/molecular mechanics simulations for the reversible phosphorylation of phosphoserine phosphatase (PSP) to study the nature of the TS. Both phosphorylation and dephosphorylation reactions are investigated based on the two-dimensional energy surfaces along phosphoryl and proton transfer coordinates. The structures of the active site at TS in both reactions reveal compact geometries, consistent with crystal structures of PSP with phosphate analogues. However, the electron density of the phosphoryl group in both TS structures slightly decreases compared with that in the reactant states. These findings suggest that the TS of PSP has a geometrically associative yet electronically dissociative character and strongly depends on proton transfer being coupled with phosphoryl transfer. Structure and literature database, which searches on phosphotransferases, suggest that such a hybrid TS is consistent with many structures and physical organic studies and likely holds for most enzymes catalyzing phosphoryl transfer.
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