Acetonitrile-induced modulation of alkaline protease as a biological macromolecule: Micro- and macro-scale effects on...

This study investigated the effects of acetonitrile as an activator solvent on the kinetic and thermodynamic properties of alkaline protease at both micro and macro levels. Micro-level effects were examined using solvatochromic parameters (π*, α, β), while macro-level effects were analyzed based on log P values and dielectric constants at varying solvent concentrations. Multiple linear regression analysis revealed the relationships between these physicochemical parameters and enzymatic behavior. Increasing acetonitrile concentrations led to decreased activation free energy (ΔG^#) and transition-state binding free energy (ΔG^#_ES), indicating the stabilization of the alkaline protease-substrate complex. A reduction in dielectric constant correlated with enhanced catalytic efficiency, as reflected by increased k_cat and k_cat/K_m values. The solvatochromic parameter π* played a key role in modulating enzyme activity through dipolarity/polarizability interactions, while β significantly influenced substrate binding affinity. Furthermore, the log P value of acetonitrile enhanced enzyme activity by reducing microenvironmental polarity, strengthening electrostatic interactions, and stabilizing the catalytic conformation. The observed relationships between solvent concentration, decreasing K_m values, and improved thermodynamic properties confirmed the activator role of acetonitrile. These findings provide novel insights into solvent-driven modulation of alkaline protease catalysis, emphasizing the interplay between micro and macro physicochemical factors.