Understanding the Rate-Limiting Step of Glycogenolysis by Using QM/MM Calculations on Human Glycogen Phosphorylase.

Liver glycogen phosphorylase (GP) is a key enzyme for human health, as its increased activity is associated with type 2 diabetes. The GP catalytic mechanism has been explored by quantum mechanics/molecular mechanics (QM/MM) methods. Herein, we propose a mechanism that proceeds by three steps: 1) it begins with transfer of a hydrogen atom from the phosphate group of the pyridoxal 5'-phosphate (HPO4 2- -PLP) cofactor to the phosphate substrate; 2) the glycosidic linkage is then cleaved through protonation of the glycosidic oxygen atom by a hydroxy group of the inorganic phosphate group; and 3) an oxygen atom of the phosphate performs a nucleophilic attack on the anomeric carbon atom of glucose, concomitant with the return of a proton from phosphate to PO4 3- -PLP, which finally leads to formation of the glucose-1-phosphate product and recovers the initial state of the PLP cofactor. The glycosidic bond cleavage and nucleophilic attack from the phosphate group to the glycosyl molecule have the highest activation free energies. The structural properties of the hereby characterized transition states could be very useful in structure-based drug design studies against liver GP.