Distal residues contribute to enzymatic catalysis in human phosphoglucose isomerase through modulation of dynamics and electrostatics

Abstract

Distal residues in enzymes, although spatially remote from the active site, play critical roles in modulating catalytic efficiency through structural dynamics and electrostatics. In this study, we investigate the contribution of distal residues to the catalytic activity of human phosphoglucose isomerase (hPGI) using molecular dynamics simulations and electrostatic calculations. Six key residues located in the second and third layers around the active site were studied previously through systematic mutagenesis. Variants, including those in the second shell K362A, Q388A, E495Q, and D511N and those in the third shell H100L and H396L, were previously reported to show significant reductions in catalytic turnover and efficiency. Analysis of structural dynamics demonstrates increased flexibility in helices critical for maintaining active site geometry, including helix-17 and helix-18, leading to destabilization of catalytic residues E358, R273, and H389′. In addition, the important interaction between H389′ and E217, essential for the ring-opening step, was disrupted across all variants. Electrostatic perturbations, including altered protonation equilibria and pKa values of catalytic residues, further impede key steps of the catalytic mechanism, such as ligand binding, ring opening, and isomerization. These findings reveal the complex interplay between distal residues, structural dynamics, and electrostatics in regulating hPGI’s catalytic mechanism, providing insights into enzyme function and guiding future enzyme engineering and drug design efforts.