Enzyme kinetics-guided reaction engineering for enhancing therapeutic enzyme substrate affinity and anti-tumor activity

Reaction engineering is key to enhance enzyme-catalyzed reactions. While previous studies have focused on boosting enzyme activity, they often overlook enzyme kinetics—especially the K m ( S 0.5 ) value—that are crucial for optimal performance under physiological conditions. Herein, we developed a novel enzyme-kinetics-guided reaction engineering strategy to improve the substrate affinity of therapeutic enzymes. In this proof-of-concept experiment, we selected arginine deiminase (ADI), a promising therapeutic enzyme for treating arginine-auxotrophic tumors. First, a kinetic trap was identified, and an enzyme-kinetics-based screening model was developed to overcome it. Next, we identified and characterized a novel arginine deiminase (ADI) from Gammaproteobacteria ( Gam ADI), which was subsequently engineered to enhance its catalytic efficiency under the guidance of the kinetic model. Through ultra-high-throughput screening, two ADI variants with low S 0.5 values were identified. After recombination, the final variant GamADI M7 exhibited a remarkable improvement in substrate affinity (91 % reduction in S 0.5 value) and a 1382-fold enhancement in catalytic efficiency. The therapeutic potential of GamADI M7 was validated via cytotoxicity assays in melanoma and prostate cancer cell lines, where its IC 50 values decreased dramatically compared to wild-type ADI. This study highlights the power of enzyme-kinetics-guided reaction engineering, offering a promising strategy to enhance enzyme substrate affinity.