Hydrolysis of acetylthiocoline, o-nitroacetanilide and o-nitrotrifluoroacetanilide by fetal bovine serum acetylcholinesterase.
Besides esterase activity, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze o-nitroacetanilides through aryl acylamidase activity. We have reported that BuChE tetramers and monomers of human blood plasma differ in o-nitroacetanilide (ONA) hydrolysis. The homology in quaternary structure and folding of subunits in the prevalent BuChE species (G4(H)) of human plasma and AChE forms of fetal bovine serum prompted us to study the esterase and amidase activities of fetal bovine serum AChE. The k(cat)/K(m) values for acetylthiocholine (ATCh), ONA and its trifluoro derivative N-(2-nitrophenyl)-trifluoroacetamide (F-ONA) were 398 x 10(6) M(-1) min(-1), 0.8 x 10(6) M(-1) min(-1), and 17.5 x 10(6) M(-1) min(-1), respectively. The lack of inhibition of amidase activity at high F-ONA concentrations makes it unlikely that there is a role for the peripheral anionic site (PAS) in F-ONA degradation, but the inhibition of ATCh, ONA and F-ONA hydrolysis by the PAS ligand fasciculin-2 points to the transit of o-nitroacetalinides near the PAS on their way to the active site. Sedimentation analysis confirmed substrate hydrolysis by tetrameric 10.9S AChE. As compared with esterase activity, amidase activity was less sensitive to guanidine hydrochloride. This reagent led to the formation of 9.3S tetramers with partially unfolded subunits. Their capacity to hydrolyze ATCh and F-ONA revealed that, despite the conformational change, the active site architecture and functionality of AChE were partially retained.
Montenegro
María F
MF
Departamento de Bioquímica y Biología Molecular-A, Universidad de Murcia, Murcia, Spain.
Moral-Naranjo
María T
MT
Muñoz-Delgado
Encarnación
E
Campoy
Francisco J
FJ
Vidal
Cecilio J
CJ
D000083:Acetanilides / Q000737:chemistry / Q000378:metabolism*; D000110:Acetylcholinesterase / Q000097:blood / Q000378:metabolism*; D000122:Acetylthiocholine / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D019791:Guanidine / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics
C539847:2-nitrotrifluoroacetanilide; D000083:Acetanilides; D000122:Acetylthiocholine; D000110:Acetylcholinesterase; D019791:Guanidine; C539848:2-nitroacetanilide