On the active site for hydrolysis of aryl amides and choline esters by human cholinesterases.
Cholinesterases, in addition to their well-known esterase action, also show an aryl acylamidase (AAA) activity whereby they catalyze the hydrolysis of amides of certain aromatic amines. The biological function of this catalysis is not known. Furthermore, it is not known whether the esterase catalytic site is involved in the AAA activity of cholinesterases. It has been speculated that the AAA activity, especially that of butyrylcholinesterase (BuChE), may be important in the development of the nervous system and in pathological processes such as formation of neuritic plaques in Alzheimer's disease (AD). The substrate generally used to study the AAA activity of cholinesterases is N-(2-nitrophenyl)acetamide. However, use of this substrate requires high concentrations of enzyme and substrate, and prolonged periods of incubation at elevated temperature. As a consequence, difficulties in performing kinetic analysis of AAA activity associated with cholinesterases have hampered understanding this activity. Because of its potential biological importance, we sought to develop a more efficient and specific substrate for use in studying the AAA activity associated with BuChE, and for exploring the catalytic site for this hydrolysis. Here, we describe the structure-activity relationships for hydrolysis of anilides by cholinesterases. These studies led to a substrate, N-(2-nitrophenyl)trifluoroacetamide, that was hydrolyzed several orders of magnitude faster than N-(2-nitrophenyl)acetamide by cholinesterases. Also, larger N-(2-nitrophenyl)alkylamides were found to be more rapidly hydrolyzed by BuChE than N-(2-nitrophenyl)acetamide and, in addition, were more specific for hydrolysis by BuChE. Thus, N-(2-nitrophenyl)alkylamides with six to eight carbon atoms in the acyl group represent suitable specific substrates to investigate further the function of the AAA activity of BuChE. Based on the substrate structure-activity relationships and kinetic studies, the hydrolysis of anilides and esters of choline appears to utilize the same catalytic site in BuChE.
Darvesh
Sultan
S
Department of Medicine (Neurology), Dalhousie University, Halifax, NS, Canada. sultan.darvesh@dal.ca
McDonald
Robert S
RS
Darvesh
Katherine V
KV
Mataija
Diane
D
Mothana
Sam
S
Cook
Holly
H
Carneiro
Karina M
KM
Richard
Nicole
N
Walsh
Ryan
R
Martin
Earl
E
D000577:Amides / Q000737:chemistry; D000581:Amidohydrolases / Q000737:chemistry; D000813:Anilides / Q000737:chemistry*; D001665:Binding Sites; D002091:Butyrylcholinesterase / Q000737:chemistry*; D002794:Choline / Q000737:chemistry; D002802:Cholinesterases / Q000737:chemistry; D017076:Computer-Aided Design; D004952:Esters / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D009574:Nitro Compounds / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000577:Amides; D000813:Anilides; D004952:Esters; D009574:Nitro Compounds; D002091:Butyrylcholinesterase; D002802:Cholinesterases; D000581:Amidohydrolases; C019732:aryl acylamidase; D002794:Choline