2.3. Mechanism of Inhibition and Enzyme Kinetics

The mechanism of enzyme inhibition was determined using the HRP-luminol coupled enzyme assay described above. Reaction velocity (max RLU/sec over 10 min) was measured across multiple substrate and inhibitor concentrations, and individual triplicate values were plotted using a Lineweaver–Burk transformation (double-reciprocal) in GraphPad Prism 9.3 software (Graph-Pad, San Diego, CA, USA). A global fit analysis was performed for competitive enzyme inhibition, which was used to calculate Ki (R² ≥ 0.98).