QM/MM reveals the sequence of substrate binding during OPRT action

Computational investigation of orotate phosphoribosyltransferase (OPRT) action, an enzymatic reaction between phosphoribosyl pyrophosphate (PRPP) and orotic acid (OA) to yield orotidine 5'-monophosphate (OMP), was carried out. Insights into the pathways of the substrate attack step of the reaction were developed under the quantum mechanics/molecular mechanics framework with S. cerevisiae strain as the representative Enzyme bearer. Four pathways were proposed for PRPP and OA binding differing in the sequence of PRPP, OA and Mg²⁺ ion complexation with OPRT. The formation of Mg²⁺-OPRT complex was accompanied by a small energy change while the largest stabilization was observed for the formation of Mg²⁺-PRPP complex supporting the experimental observation of Mg²⁺-PRPP complex as the true substrate for the reaction. Formation of PRPP-OPRT complex was found to be energetically not probable rendering the pathway requiring Mg²⁺-OA complex not probable. Further, PRPP migration towards the active site was found to be energetically not favoured rendering the pathway involving Mg²⁺-OA complexation improbable. Migration of OA and Mg²⁺-PRPP complex towards the active site was found to be energetically probable with a large stabilization of the system when Mg²⁺-PRPP complex bound to the OA-OPRT complex. This conclusively proved the sequential binding of OA and Mg²⁺-PRPP complexes during OPRT action.