title: Kinetic analysis of butyrylcholinesterase-catalyzed hydrolysis of acetanilides.
abstract: The aryl-acylamidase (AAA) activity of butyrylcholinesterase (BuChE) has been known for a long time. However, the kinetic mechanism of aryl-acylamide hydrolysis by BuChE has not been investigated. Therefore, the catalytic properties of human BuChE and its peripheral site mutant (D70G) toward neutral and charged aryl-acylamides were determined. Three neutral (o-nitroacetanilide, m-nitroacetanilide, o-nitrophenyltrifluoroacetamide) and one positively charged (3-(acetamido) N,N,N-trimethylanilinium, ATMA) acetanilides were studied. Hydrolysis of ATMA by wild-type and D70G enzymes showed a long transient phase preceding the steady state. The induction phase was characterized by a hysteretic "burst". This reflects the existence of two enzyme states in slow equilibrium with different catalytic properties. Steady-state parameters for hydrolysis of the three acetanilides were compared to catalytic parameters for hydrolysis of esters giving the same acetyl intermediate. Wild-type BuChE showed substrate activation while D70G displayed a Michaelian behavior with ATMA as with positively charged esters. Owing to the low affinity of BuChE for amide substrates, the hydrolysis kinetics of neutral amides was first order. Acylation was the rate-determining step for hydrolysis of aryl-acetylamide substrates. Slow acylation of the enzyme, relative to that by esters may, in part, be due suboptimal fit of the aryl-acylamides in the active center of BuChE. The hypothesis that AAA and esterase active sites of BuChE are non-identical was tested with mutant BuChE. It was found that mutations on the catalytic serine, S198C and S198D, led to complete loss of both activities. The silent variant (FS117) had neither esterase nor AAA activity. Mutation in the peripheral site (D70G) had the same effect on esterase and AAA activities. Echothiophate inhibited both activities identically. It was concluded that the active sites for esterase and AAA activities are identical, i.e. S198. This excludes any other residue present in the gorge for being the catalytic nucleophile pole.
authors: Centre de Recherches du Service de Santé des Armées, Département de Toxicologie, Unité d'Enzymologie, BP 87, 38702 La Tronche cedex, France. pymasson@compuserve.com
authors: Patrick
authors: P
authors: Masson
authors: Marie-Thérèse
authors: MT
authors: Froment
authors: Emilie
authors: E
authors: Gillon
authors: Florian
authors: F
authors: Nachon
authors: Sultan
authors: S
authors: Darvesh
authors: Lawrence M
authors: LM
authors: Schopfer
mesh_terms: D000083:Acetanilides / Q000378:metabolism*; D000581:Amidohydrolases / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D002091:Butyrylcholinesterase / Q000235:genetics / Q000378:metabolism*; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D004456:Echothiophate Iodide / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D020836:Protein Structure, Quaternary; D013237:Stereoisomerism
chemical_list: D000083:Acetanilides; D002800:Cholinesterase Inhibitors; D004456:Echothiophate Iodide; D002091:Butyrylcholinesterase; D000581:Amidohydrolases; C019732:aryl acylamidase