Dynamic protonation states and vacancy migration modulate glutaminase C catalysis: essential role of Lys481 and Tyr466

Abstract

Computer modelling of the enzyme-substrate complex structure of the glutaminase C (GAC) filament was performed using molecular dynamics (MD) calculations with classical and combined (quantum mechanics/molecular mechanics, QM/MM) potentials. The modelling results demonstrate the necessity of a proton vacancy in the protein’s active site for the nucleophilic attack of the key residue Ser286 side chain on the substrate. The relative stability of various protonated states of amino acid residues in the active site of the protein-ligand complex was evaluated. It was revealed that the proton vacancy is most likely located on the amino group of the Lys481 side chain, but protons readily migrate between ionizable groups of the active site residues, facilitating the nucleophilic attack by enabling deprotonation of the serine residue side chain. The constructed models also explain the absence of catalytic activity in the GAC variant with the Tyr466Trp substitution, which is caused by Trp466 displacing the Lys481 side chain away from the active site.