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 (diisopropylphosphoryl-BuChE (DIP-BuChE)) was investigated. Hydrostatic pressure strongly increased the rate of aging of wt enzyme. The activation volumes (deltaV*) for the dealkylation reaction was -150 ml/mol for DIP-wtBuChE. On the other hand, pressure had little effect on the aging of the DIP-E197D mutant and no effect on the DIP-D70G mutant, indicating that the transition state of the aging reaction (dealkylation of an isoproxy chain) was associated with an extended conformation/hydration change in wtBuChE but not in mutants. The rate of aging decreased with osmotic pressure, supporting the idea that water is important for stabilizing the transition state. Molecular dynamics simulations were performed on the wtDIP adduct to relate the kinetic data to hydration changes in the enzyme active site gorge. The pH dependence of the melting temperature (Tm) of native and soman-wtBuChE, as determined by differential scanning calorimetry (DSC), indicated that the stabilization energy of aged BuChE is mainly due to the salt bridge between protonated H438 and PO-, with pK(H438) = 8.3. Electrophoresis under high pressure up to 2.5 kbar showed that aged wtBuChE did not undergo pressure-induced molten globule transition unlike the native enzyme. This transition was not seen for the mutant enzymes, indicating that mutants are resistant 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 network dynamics in the active site gorge of BuChE, both residues acting like valves. In mutant enzymes, mutated residues function like check valves: forced penetration of water in the gorge is difficult, release of water is facilitated.
Masson
P
P
Centre de Recherches du Service de Sante des Armees, Unite d'Enzymologie, La Tronche, France. 100335.404@compuserve.com
Fortier
P L
PL
Albaret
C
C
Cléry
C
C
Guerra
P
P
Lockridge
O
O
D000375:Aging / Q000378:metabolism; D001665:Binding Sites; D002091:Butyrylcholinesterase / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D009997:Osmotic Pressure; D010766:Phosphorylation; D011489:Protein Denaturation; D012999:Soman / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D002800:Cholinesterase Inhibitors; D007531:Isoflurophate; D012999:Soman; D002091:Butyrylcholinesterase