title: Photoreversible inhibition of cholinesterases: catalytic serine-labeled caged butyrylcholinesterase.
abstract: The photoregulation of the catalytic activity of butyrylcholinesterase (BChE) was investigated by treating the enzyme with a newly developed carbamylating reagent, N-methyl-N-(2-nitrophenyl)carbamoyl chloride, which has proved to be an efficient photoremovable alcohol-protecting group. BChE was efficiently inhibited in a time- and concentration-dependent manner, and the enzyme could be protected against inhibition by active-site-specific ligands (that is, tacrine). The inactivation kinetics showed a biphasic character, which can be analyzed as the result of the existence of two conformational states in solution. Pseudo-irreversible inactivation of BChE, which results from catalytic serine carbamylation, was suggested by recovery of the enzyme activity after dilution and was demonstrated by X-ray crystallography. Remarkably, the 3D structure of the carbamylated BChE conjugate showed a nonambiguous carbamylation of the catalytic serine residue as the only chemical modification on the protein. The photoreversibility of the enzyme inactivation was analyzed by irradiating the inactivated enzyme at 365 nm and was shown to reach completion in some conditions. The efficient and specific "caging" of BChE, together with the availability of carbamylated BChE crystals, will offer a unique possibility to study the catalytic properties of this enzyme by kinetic crystallography after cryophotolytic uncaging of the enzyme conjugate crystals.
authors.affiliation: Laboratoire de Chimie Bioorganique, UMR 7514 CNRS, Faculté de Pharmacie, Université Louis Pasteur Strasbourg, B.P. 24, 67401 Illkirch Cedex, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Loudwig
authors.forename: Yvain
authors.initials: Y
authors.lastname: Nicolet
authors.forename: Patrick
authors.initials: P
authors.lastname: Masson
authors.forename: Juan C
authors.initials: JC
authors.lastname: Fontecilla-Camps
authors.forename: Suzanne
authors.initials: S
authors.lastname: Bon
authors.forename: Florian
authors.initials: F
authors.lastname: Nachon
authors.forename: Maurice
authors.initials: M
authors.lastname: Goeldner
mesh_terms: D000818:Animals; D001665:Binding Sites; D002091:Butyrylcholinesterase; D016466:CHO Cells; D002384:Catalysis; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D006224:Cricetinae; D018360:Crystallography, X-Ray; D004793:Enzyme Reactivators / Q000494:pharmacology*; D006801:Humans; D010782:Photolysis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000737:chemistry*; D013619:Tacrine / Q000494:pharmacology
chemical_list: D002800:Cholinesterase Inhibitors; D004793:Enzyme Reactivators; D011994:Recombinant Proteins; D012694:Serine; D013619:Tacrine; D002091:Butyrylcholinesterase