Stojan Jure, Brochier Laure, Alies Carole, Colletier Jacques Philippe, Fournier Didier
Institute of Biochemistry, Medical Faculty, University of Ljubljana, Slovenia.
Eur J Biochem. 2004 Apr;271(7):1364-71. doi: 10.1111/j.1432-1033.2004.04048.x.
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 binds at the rim 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) when the 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 active-site before the catalytic serine is deacetylated, its new position will prevent the approach of water, thus blocking deacetylation.
在高底物浓度下，乙酰胆碱酯酶对乙酰胆碱的水解受到抑制。为了确定参与这一现象的残基，我们对活性位点峡谷周围的大多数残基进行了突变，但这些突变并没有完全消除水解作用。因此，我们分析了一种不可水解的底物类似物对底物水解以及对乙酰化酶类似物再活化的影响。对各种模型的分析使我们提出了以下一系列事件：底物最初结合在活性位点峡谷的边缘，然后滑向峡谷底部并在那里被水解。另一个底物分子可以结合到外周位点：(a)当胆碱仍在峡谷内时，它会阻碍胆碱的排出；(b)胆碱解离后但在脱乙酰化发生之前，在外周位点的结合会提高脱乙酰化速率，但是(c)如果结合到外周位点的底物分子在催化丝氨酸脱乙酰化之前滑到活性位点底部，其新位置会阻止水的接近，从而阻断脱乙酰化。
