Existing data show that the initial phases of inactivation of serine hydrolase enzymes by organophosphorus compounds resemble those of acylation. The thesis of this proposal is that the characteristic differences between carbon and phosphorus in valence and electronic structure dominate the ensuing processes. An investigation of these later phases, the stabilizing forces in the adducts and the dynamics of their decomposition can reveal much about the scope of the catalytic potential of serine hydrolases and how enzyme regeneration might be promoted over aging. Reversible phosphonate ester inhibitors of serine hydrolases might be used as blocking agents in pretreatment against soman and sarin. The specific goal of this project is the modeling of interactions between fully hydrated trypsin, chymotrypsin or acetylcholinesterase and organophosphorus inhibitors: 2-propyl and pinacolyl methyphosphonofluoridates, 4-nitrophenyl 4- substituted-phenacyl methylphosphonates. Charge distribution will be calculated with program MNDO and nonbonded interactions will be evaluated in the course of adduct formation and in the diastereomeric adducts by molecular graphics and molecular mechanics calculations using program YETI. The effect of positive charge on His-57, when each N is protonated, will also be studies as a potential stabilizing factor in the serine hydrolase-organophosphate adducts. Isotopic probes and NMR measurements will also be employed for the study of the mechanism of the general acid and base catalyzed aging reactions. These and results of previous investigations of the kinetics of phosphonylation dephosphonylation and enzymic dealkylation in the phosphonate ester adducts will then be correlated with calculated energies and geometries in the adducts. The factors promoting dephosphonylation and disfavor aging will then be assessed. %%% The results of this experiment will contribute to an understanding of the stereochemical outcome of serine protease phosphonylation, thereby furthering knowledge regarding the modes of interaction at the catalytic site. From the practical point of view, if one could learn the key elements involved in the stability and dynamic of adducts, one could promote dephosphonylation at will and thereby design inhibitors with controlled reversibility. Ideas of this kind can find use in medical application and in protein synthesis by molecular engineering.
