Structural and hydration changes in the active site gorge of phosporhylated butyrylcholinesterase accompanying the aging process

Wild-type (wt) butyrylcholinesterase (BuChE) and the E197D and D70G mutants were inhibited by diisopropylfluorophosphate (DFP) or soman under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process of DFP-phosphorylated enzymes (diiso propylphosphoryl-BuChE (DIP-BuChE)) was investigated. Hydrostatic pressure strongly increased the rate of aging of wt enzyme. The activation volumes ( Delta V-not equal) for the dealkylation reaction was -150 ml/mol for DIP-wt -BuChE. On the other hand, pressure had little effect on the aging of the D IP-E197D mutant and no effect on the DIP-D70G mutant, indicating that the t ransition state of the aging reaction (dealkylation of an isoproxy chain) w as associated with an extended conformation/ hydration change in wtBuChE bu t not in mutants. The rate of aging decreased with osmotic pressure, suppor ting the idea that water is important for stabilizing the transition state. Molecular dynamics simulations were performed on the wtDIP adduct to relat e the kinetic data to hydration changes in the enzyme active site gorge. Th e pH dependence of the melting temperature (T-m) of native and soman-wtBuCh E, as determined by differential scanning calorimetry (DSC), indicated that the stabilization energy of aged BuChE is mainly due to the salt bridge be tween protonated H438 and PO-, with PKH438 = 8.3. Electrophoresis under hig h pressure up to 2.5 kbar showed that aged wtBuChE did not undergo pressure -induced molten globule transition unlike the native enzyme. This transitio n was not seen for the mutant enzymes, indicating that mutants are resistan t to the penetration of water into their structure. Our results support the conclusion that D70 and E197 are major residues for the water/H-bond netwo rk dynamics in the active site gorge of BuChE, both residues acting like va lves. In mutant enzymes, mutated residues function like check valves: force d penetration of water in the gorge is difficult, release of water is facil itated. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.