ИСТИНА

Reactibody A17 was selected from Griffin library against arylphosphonate. The deep structural and functional analysis has revealed the architecture of its active center to have a cholinesterase-like anion binding site, hydrophobic pocket, and reactive Tyr residue. Reactibody A17 showed high reactivity with parent substrate, an aryl-phosphonate. As expected, the reaction was noncatalytic. We found that reactibody A17 does not react with echothiophate and p-nitrophenyl phosphocholine, but does interact with DFP, AEBSF and paraoxon. Moreover we observed paraoxon hydrolysis by A17 and showed that this process involves covalent intermediate formation. Although our selection method was designed primarily to provide covalent binding, our general task remains to obtain catalytic antibodies. To that end, we have identified one substrate which is hydrolyzed with catalytic turnover. We have established that the for initial non-covalent binding and proper orientation, the positive amino acids is preferred, the hydrolysis require the negative charged amino acids for stabilization of hydroxyl ion and decreasing of proton transfer. To engineer an abzyme based on the A17 reactibody to turn over organophosphorus substrates we used two approaches of active center evolution. We created the sub-library of reactibody active center and developed the computational maturation to provide best driving forces for paraoxon hydrolyzing antibody. We confirmed that arginine mutant showed high reactivity with paraoxon. It was two orders of magnitude more compared to wild type. But no catalysis was observed. Thus our results were in line with our computed predictions. A histidine-35 mutant improved covalent binding with paraoxon and retained same catalytic activity. Glutamic acid-35 mutant blocked interaction with both phosphonate and paraoxon molecules.