Electrostatic Preorganization and Mg2+ Positioning in β-Phosphoglucomutase

Catalytic activity in β-phosphoglucomutase (β-PGM) has previously been attributed to early proton transfer from the neighboring Asp10 residue to the leaving phosphoryl oxygen during catalysis. Here, we revisit this mechanistic interpretation by quantifying the electrostatic environment of wild-type (WT) β-PGM and the Asp10Asn mutant using AMOEBA polarizable molecular dynamics simulations combined with electric-field analysis. We computed projected electric fields along phosphoryl- and proton-transfer coordinates in both the ground state (GS) and a transition-state/product-state (TS–PS) hybrid, and decomposed these fields into residue-, ion-, and solvent-specific contributions. We find that the WT enzyme is electrostatically preorganized in the GS to support phosphoryl transfer and to make the Asp10 proton labile, but the projected fields do not indicate that this proton is directly driven onto the substrate. In the Asp10Asn mutant, this early preorganization is lost and only partially compensated in the TS–PS. Importantly, we find that the dominant disruption in the Asp10Asn mutant is the reorganization of Mg2+: rather than maintaining productive phosphoryl-transfer alignment as in the WT, Mg2+ becomes mispositioned in the mutant and strongly opposes phosphoryl transfer in the GS. Water provides some late-stage compensation in the mutant TS–PS, but it does not restore the missing GS electrostatic preorganization.