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


Abstract

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. Study holds ProTherm entries: 12944, 12945 Extra Details: butyrylcholinesterase; organophosphate inhibitor; aging; hydorophobic pressure;,osmotic pressure; molecular dynamics

Submission Details

ID: JsBfRcXq

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:44 p.m.

Version: 1

Publication Details
Masson P;Fortier PL;Albaret C;Cléry C;Guerra P;Lockridge O,Chem. Biol. Interact. (1999) Structural and hydration changes in the active site gorge of phosporhylated butyrylcholinesterase accompanying the aging process. PMID:10421435
Additional Information

Study Summary

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
6EQP 2017-10-14T00:00:00+0000 2.35 Human butyrylcholinesterase in complex with ethopropazine
6EQQ 2017-10-15T00:00:00+0000 2.4 Human butyrylcholinesterase in complex with huprine 19
6RUA 2019-05-27T00:00:00+0000 2.75 Structure of recombinant human butyrylcholinesterase in complex with a coumarin-based fluorescent probe linked to sulfonamide type inhibitor.
4AXB 2012-06-12T00:00:00+0000 2.4 Crystal structure of soman-aged human butyrylcholinesterase in complex with 2-PAM
5DYY 2015-09-25T00:00:00+0000 2.65 Crystal structure of human butyrylcholinesterase in complex with N-((1-benzylpiperidin-3-yl)methyl)naphthalene-2-sulfonamide
6T9S 2019-10-28T00:00:00+0000 2.7 Human Butyrylcholinesterase in complex with 2-(N-hydroxyimino)-N-[(1S)-3-{4-[(2-methyl-1H-imidazol-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1- phenylpropyl]acetamide
5DYT 2015-09-25T00:00:00+0000 2.55 Crystal structure of human butyrylcholinesterase in complex with N-((1-benzylpiperidin-3-yl)methyl)-N-methylnaphthalene-2-sulfonamide
3DKK 2008-06-25T00:00:00+0000 2.31 Aged Form of Human Butyrylcholinesterase Inhibited by Tabun
2XQI 2010-09-02T00:00:00+0000 2.6 X-ray Structure of human butyrylcholinesterase inhibited by racemic CVX
2XQG 2010-09-02T00:00:00+0000 2.3 X-ray Structure of human butyrylcholinesterase inhibited by racemic VR

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.3 BCHE protein P21927 CHLE_RABIT
95.0 BCHE protein P81908 CHLE_HORSE
92.1 BCHE protein O62761 CHLE_PANTT
92.1 BCHE protein O62760 CHLE_FELCA
92.1 BCHE protein P32749 CHLE_BOVIN
100.0 BCHE protein P06276 CHLE_HUMAN