Mechanism of Mg²⁺-Accompanied Product Release in Sugar Nucleotidyltransferases

The nucleotidyl transfer reaction, catalyzed by sugar nucleotidyltransferases (SNTs), is assisted by two active site Mg²⁺ ions. While studying this reaction using X-ray crystallography, we captured snapshots of the pyrophosphate (product) as it exits along a pocket. Surprisingly, one of the active site Mg²⁺ ions remains coordinated to the exiting pyrophosphate. This hints at the participation of Mg²⁺ in the process of product release, besides its role in catalyzing nucleotidyl transfer. These observations are further supported by enhanced sampling molecular dynamics simulations. Free energy computations suggest that the product release is likely to be rate limiting in SNTs, and the origin of the high free energy barrier for product release could be traced back to the "slow" conformational change of an Arg residue at the exit end of the pocket. These results establish a dual role for Mg²⁺, and propose a general mechanism of product release during the nucleotidyl transfer by SNTs.