tailieunhanh - Báo cáo khoa học: Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate

Acetylcholine hydrolysis by acetylcholinesterase is inhi-bited at high substrate concentrations. To determine the residues involved in this phenomenon, we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus, we analyzed the effect of a nonhydrolysable sub-strate analogue on substrate hydrolysis and on reactiva-tion of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events:the substrate initially binds at the rim of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed | Eur. J. Biochem. 271 1364-1371 2004 FEBS 2004 doi Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate Jure Stojan1 Laure Brochier2. Carole Alies2 Jacaues PhiliDDe Colletier2 and Didier Fournier2 1 Institute of Biochemistry Medical Faculty University of Ljubljana Slovenia 2IPBS-UMR 5089 Toulouse France Acetylcholine hydrolysis by acetylcholinesterase is inhibited at high substrate concentrations. To determine the residues involved in this phenomenon we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus we analyzed the effect of a nonhydrolysable substrate analogue on substrate hydrolysis and on reactivation of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events the substrate initially butcls at the im of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed. Another substrate molecule can bind to the peripheral site a wlnn die choline is still inside the gorge - it will thereby hinder its exit b after choline has dissociated but before deacetylation occurs - binding at the peripheral site increases deacetylation rate but c if a substrate molecule bound to the peripheral site slides down to the bottom of the activesite before the catalytic serine is deacetylated its new position will prevent the approach of water thus blocking deacetylation. Keywords acetylchoiẽ s deacyladon ínhibitíon kín-etics structure-function. Cholinesterases acetylcholinesterases AChEs and butyrylcholinesterases are serine hydrolases that hydrolyze choline esters in two steps tytakitiwi of ihe en yme. followed by deacylation involving a water molecule 1 . In the case of AChEs the process takes place at the acetylation site located at the bottom of a 20 A-deep gorge usually called the active-site gorge. The site includes a tryptophan residue that .