Pentacoordinated phosphorus revisited by high‐level QM/MM calculations

Enzymes catalyzing phosphoryl transfer reactions are extremely efficient and are involved in crucial biochemical processes. The mechanisms of these enzymes are complex due to the diversity of substrates that are involved. The reaction can proceed through a pentacoordinated phosphorus species that is either a stable intermediate or a transition state (TS). Because of this, the first X‐ray structure of a pentacoordinated phosphorus intermediate in the β‐phosphoglucomutase enzyme aroused great interest but also much controversy. To provide new insights into the nature of that structure, we have determined the reaction path of the phosphorylation step using high‐level QM/MM calculations, and have also calculated the geometry of a complex with a transition state analogue (TSA) that has been suggested to be the actual species in the crystal. The protein crystalline environment has been modeled so as to mimic the experimental conditions. We conclude that the pentacoordinated phosphorus formed in this enzyme is not a stable species but a TS, which gives an activation energy for phosphorylation in agreement with kinetic results. We also show that the TSA is a good mimic of the true TS. We have performed a new crystallographic refinement of the original diffraction map of the pentacoordinated phosphorus structure with the MgF TSA. The new fit improves significantly with respect to the original one, which strongly supports that Allen and coworkers wrongly assigned the X‐ray structure to a pentavalent phosphorane. Proteins 2010. © 2010 Wiley‐Liss, Inc.