Semi-Rational Engineering of rhArg1 via Non-Conserved Residue Mutagenesis for Enhanced Efficiency and Stability

Arginase 1 deficiency is a rare inherited metabolic disorder characterized by progressive neurological dysfunction. Although recombinant human arginase 1 (rhArg1) has been investigated as a therapeutic enzyme, its clinical potential is limited by insufficient catalytic activity and stability. In this study, we applied an evolutionary conservation-guided design strategy to enhance rhArg1 activity by targeting low-conservation residues within the catalytic pocket. Seven candidate sites were identified, and 29 variants were generated by site-directed and combinatorial mutagenesis. Under 2 mM Mn²⁺ conditions, several mutants (N139K, Q143K, and Q143S) exhibited 1.4-1.6 fold increases in kcat relative to the wild type. Notably, under Mn²⁺-free conditions, the double mutant T136D/Q143V showed a 1.5-fold increase in kcat and a 73% improvement in kcat/KM. Molecular dynamics simulations revealed that these mutations enhance catalytic performance by promoting near-attack conformations. This work demonstrates an effective rational strategy for improving rhArg1 catalytic efficiency and stability, supporting its therapeutic development.