Catalytic mechanism of 6-phosphogluconate dehydrogenase: a theoretical investigation

Density functional calculations are employed to theoretically explore the mechanism of all elementary reaction steps involved in the catalytic reaction of 6-phosphogluconate dehydrogenase (6PGDH). The model systems we choose for the enzyme contain the essential parts of the cofactor (NADP+), the substrate 6-phosphogluconate (6PG), and some key residues (Lys183 and Glu190) in the active site of sheep liver 6PGDH. The effect of the apoenzyme electrostatic environment on the studied reaction is treated by the self-consistent reaction-field method. Our calculations demonstrate that the first step of the catalytic reaction is the formation of a 3-keto 6PG intermediate, which proceeds through a concerted transition state involving a hydride transfer from 6PG to NADP+, and a proton transfer from 6PG to Lys183. The second step is the elimination of a CO2 molecule from 6-PG, concomitant with a proton transfer from Lys183 to 6-PG. In the final step, a concerted double proton transfer (one from Glu190 to the substrate, another from the substrate to Lys183) results in the final product, the keto form of ribulose 5-phosphate (Ru5P). The rate-limiting step is the formation of a 3-keto 6PG intermediate, with a free energy barrier of 22.7 kcal/mol at room temperature in the protein environment, and all three steps are calculated to be thermodynamically favorable. These results are in good agreement with the general acid/general base mechanism suggested from previous experiments for the 6PGDH reaction.