CHARACTERIZING THE ENZYME KINETICS OF NOVEL ANTIOXIDANT PATHWAYS IN PLANT CELLS UNDER STRESS CONDITIONS

Abstract

Reactive oxygen species (ROS) overproduction under drought and salinity stress threatens plant cellular homeostasis by damaging proteins, lipids, and nucleic acids. To elucidate the dynamic performance of novel antioxidant defenses, we isolated peroxiredoxin Q (PrxQ), mitochondrial thioredoxin h (TrxH), and glutaredoxin C (GrxC) from Arabidopsis leaves subjected to controlled drought and salinity treatments. Using spectrophotometric assays across varying substrate concentrations, pH (6.5–8.0), and temperatures (20–30 °C), we determined Michaelis–Menten constants (Kₘ), turnover numbers (kₖₐₜ), and catalytic efficiencies (kₖₐₜ/Kₘ). Under drought, PrxQ’s Kₘ decreased from 50 to 30 µM while kₖₐₜ rose from 100 to 120 s⁻¹, doubling efficiency to 4.0×10⁶ M⁻¹·s⁻¹; salinity induced more moderate shifts (Kₘ = 40 µM, kₖₐₜ = 110 s⁻¹, efficiency = 2.75×10⁶ M⁻¹·s⁻¹). TrxH showed a 10% reduction in Kₘ (100→90 µM) and a 12.5% increase in kₖₐₜ (80→90 s⁻¹) under drought (efficiency = 1.0×10⁶ vs. 0.8×10⁶ M⁻¹·s⁻¹ control), whereas salinity slightly impaired its kinetics. GrxC exhibited the highest drought‐induced enhancement (Kₘ 20→15 µM; kₖₐₜ 50→60 s⁻¹; efficiency 4.0×10⁶ M⁻¹·s⁻¹). PrxQ achieved optimal affinity and turnover at pH 7.5 under drought, and TrxH peaked in activity at 30 °C. All efficiency changes were statistically significant (ANOVA, p < 0.01). These results demonstrate that drought stress more strongly augments the catalytic machinery of key antioxidant pathways than salinity, offering quantitative parameters for integration into redox network models. Our findings provide a mechanistic foundation for targeted genetic or biotechnological enhancement of plant stress resilience.