Human phosphoglucose isomerase (hPGI) catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate, an important step in glycolysis. It serves also as a biomarker for certain types of cancer. Electrostatics-based computational methods THEMATICS and POOL, developed by us, predicted that several residues in the second and third layers beyond the reacting substrate molecule were active in catalysis. These predictions were confirmed by direct biochemical assay, showing that the hPGI second-shell variants K362A, Q388A, E495Q, and D511N, and the third-shell variants H396L and H100L, all show significant loss in catalytic efficiency compared to wild type. K362 is located 11 Å from the ligand; the K362A variant shows no detectable catalytic activity. Observed reductions in kcat/KM for Q388A, E495Q, and D511N in the second shell are 19-, 250-, and 230- fold, respectively; corresponding reductions for H396L and H100L in the third shell are 24- and 630- fold, respectively. Using protein electrostatics and molecular dynamics simulations, we explore the roles that these distal residues play in the catalytic process. We show that electrostatic interactions are partially responsible for these longer-range effects, in which the distal residues influence the electric field within the active site and the proton transfer properties of the first-shell catalytic residues. We also identify conformational dynamical processes that participate in the catalytic mechanism.

Computational studies of the role of distal residues in human phosphoglucose isomerase catalysis

256th National Meeting (2018)

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