ИСТИНА

Recently we developed a potent bioscavenger abzyme against organophosphates, A17­L­L47K. However, to further advance its capabilities by turning it into a full­ cycle organophosphatase, a mechanism to reprotonate catalytic tyrosine must be introduced. Taking inspiration from tyrosine phosphatases, we introduced H­A106E substitution to act as a general base. Multiscale modeling demonstrated stable pairing of E106 with the catalytic tyrosine as well as its ability to acquire and retain the tyrosine's proton until the reactivation stage. What is more, the calculated free energy barrier of the inhibition stage of 16 kcal/mol is the lowest of all A17 variants constructed to date, highlighting the importance of a well­behaved catalytic diad in bioscavenger design. We performed multiple Rosetta­powered design iterations to further stabilize the diad and optimize substrate binding. The resulting variants demonstrated a potent ability to bind paraoxon in a productive pre­reaction conformation in silico in addition to low reaction barrier and proton retention. Therefore, our work not only produced promising bioscavenger variants but also paved the way for the future design of a full­cycle abzyme organophosphatase.