Conformational plasticity of butyrylcholinesterase as revealed by high pressure experiments.
The ligand binding and kinetic behaviour of butyrylcholinesterase (EC 3.1.1.8, acylcholine acylhydrolase) from human plasma was studied at 35 degrees C under high hydrostatic pressure. The binding of phenyltrimethylammonium was studied by affinity electrophoresis at various pressures ranging from 10(-3) to 2 kbar. The kinetics of enzyme carbamylation with N-methyl(7-dimethylcarbamoxy)quinolinium iodide was studied in single-turnover conditions up to 1.2 kbar using a high-pressure stopped-flow fluorimeter. Experiments were carried out in different media: 1 mM Tris-HCl (pH 8) with water, water containing 0.1 M lithium chloride and deuterium oxide as solvents. The volume changes (delta V and delta V++) associated with each process were determined from the pressure-dependence of the binding and kinetic constants. Kinetic data show that the binding of substrate to the enzyme leads to a pressure-sensitive enzyme conformational state which cannot accomplish the catalytic act. The pressure-induced inhibitory effect is highly cooperative; it depends on both the nature (charged or neutral) and the concentration of the substrate. Also, large solvent effects indicate that enzyme sensitivity to pressure depends on the solvent structure. This findings suggests that the substrate-dependent pressure effect is modulated by the solvation state of the enzyme.
Masson
P
P
Centre de Recherches du Service de Santé des Armées, La Tronche, France.
Balny
C
C
D002091:Butyrylcholinesterase / Q000097:blood / Q000737:chemistry*; D002219:Carbamates / Q000737:chemistry; D004586:Electrophoresis; D006801:Humans; D006874:Hydrostatic Pressure; D007700:Kinetics; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D011806:Quinolinium Compounds / Q000737:chemistry
D002219:Carbamates; D000644:Quaternary Ammonium Compounds; D011806:Quinolinium Compounds; C034576:7-(dimethylcarbamoyloxy)-N-methylquinolinium; C009571:phenyltrimethylammonium; D002091:Butyrylcholinesterase