pH Regulates Ligand Binding to an Enzyme Active Site by Modulating Intermediate Populations

Area of Science:

• Biochemistry and Molecular Modeling
• Computational Chemistry
• Enzyme Kinetics

Background:

• Ligand-protein interactions are crucial for drug discovery.
• Solution pH is a key factor influencing binding thermodynamics.
• Enzymes with pH-sensitive active sites are important drug targets.

Purpose of the Study:

• To investigate the effect of pH on guanidinium ion (Gdm+) binding to hen egg-white lysozyme (HEWL).
• To elucidate the binding mechanisms and kinetics of Gdm+ at the HEWL active site.
• To provide insights for designing drugs targeting enzymes with acidic residues.

Main Methods:

• Molecular dynamics simulations were employed to study Gdm+ binding to HEWL.
• Free energy surface (FES) calculations mapped binding pathways and intermediates.
• Markov state modeling quantified kinetic pathways and state lifetimes.
• Analysis of residue protonation states in response to pH changes.

Main Results:

• Gdm+ binds to the HEWL active site via two dominant pathways with multiple intermediates.
• Active site residues play a critical role in anchoring the ligand.
• Ligand-binding rates exhibit a sharp increase near the pKa of active site acidic residues.
• Up to three Gdm+ ions can bind, with one acting as a scaffold.

Conclusions:

• pH-dependent protonation of acidic residues critically modulates Gdm+ binding to HEWL.
• Understanding these pH-dependent binding dynamics is essential for rational drug design.
• The findings support the development of novel Gdm+-containing molecules for enzyme inhibition.