Hydration change during the aging of phosphorylated human butyrylcholinesterase: importance of residues aspartate-70 and glutamate-197 in the water network as probed by hydrostatic and osmotic pressures

Wild-type human butyrylcholinesterase (BuChE) and Glu-197 --> Asp and Asp70 --> Gly mutants (E197D and D70G respectively) were inhibited by di-isoprop yl phosphorofluoridate under standard conditions of pH, temperature and pre ssure. The effect of hydrostatic:and osmotic pressures on the aging process (dealkylation of an isopropyl chain) of phosphorylated enzymes [di-isoprop ylated (DIP)-BuChE] was investigated. Hydrostatic pressure markedly increas ed the rate of aging of wild-type enzyme. The average activation volume (De lta V-not equal) for the dealkylation reaction was -170 ml/mol for DIP wild -type BuChE. On the other hand, hydrostatic pressure had little effect on t he aging of the DIP mutants (Delta V-not equal = -2.6 ml/mol for E197D and -2 ml/mol for D70G), suggesting that the transition state of the aging proc ess was associated with an extended hydration and conformational change in wild-type BuChE, but not in the mutants. The rate of aging of wild-type and mutant enzymes decreased with osmotic pressure, allowing very large positi ve osmotic activation volumes (Delta V-osm(not equal)) to be estimated, thu s probing the participation of water in the aging process. Molecular dynami cs simulations performed on the active-site gorge of the wild-type DIP addu ct showed that the isopropyl chain involved in aging was highly solvated, s upporting the idea that water is important for stabilizing the transition s tate of the dealkylation reaction. Wild-type BuChE was inhibited by soman ( pinacolyl methylphosphonofluoridate). Electrophoresis performed under high pressure [up to 2.5 kbar (1 bar = 10(5) Pa)] showed that the soman-aged enz yme did not pass through a pressure-induced, molten-globule transition, unl ike the native wild-type enzyme. Likewise, this transition was not seen for the native E197D and D70G mutants, indicating that these mutants are resis tant to the penetration of water into their structure. The stability energe tics of native and soman-aged wild-type BuChE were determined by differenti al scanning calorimetry. The pH dependence of the midpoint transition tempe rature of endotherms indicated that the high difference in stabilization en ergy between aged and native BuChE (Delta Delta G = 23.7 kJ/mol at pH 8.0) is mainly due to the salt bridge between protonated His-438 and PO-, with p K(His-438) = 8.3. A molecular dynamics simulation on the MIP adduct showed that there is no water molecule around the ion pair. The 'hydrostatic versu s osmotic pressure' approach probed the importance of water in aging, and a lso revealed that Asp-70 and Glu-197 are the major residues controlling bot h the dynamics and the structural organization of the water/hydrogen-bond n etwork in the active-site gorge of BuChE. In wild-type BuChE both residues function like valves, whereas in the mutant enzymes the water network is sl ack, and residues Gly-70 and Asp-197 function like check valves, i.e. force d penetration of water into the gorge is not easily achieved, thereby facil itating the release of water.