Colletier Jacques-Philippe, Fournier Didier, Greenblatt Harry M, Stojan Jure, Sussman Joel L, Zaccai Giuseppe, Silman Israel, Weik Martin
Laboratoire de Biophysique Moléculaire, Institut de Biologie Structurale (CEA/CNRS/UJF), Grenoble Cedex, France.
EMBO J. 2006 Jun 21;25(12):2746-56. doi: 10.1038/sj.emboj.7601175. Epub 2006 Jun 8.
Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.
乙酰胆碱酯酶（AChE）通过快速水解神经递质乙酰胆碱来终止胆碱能突触处的神经冲动传递。AChE中的底物转运涉及至少两个结合位点，即催化位点和外周阴离子位点，这两个位点被认为存在变构关系并参与底物抑制作用。在此，我们展示了加州电鳐AChE与底物乙酰硫代胆碱、产物硫代胆碱以及一种不可水解的底物类似物形成的复合物的晶体结构。这些结构提供了底物到达活性位点途中的一系列静态快照，并首次确定了底物和产物在外周位点及活性位点的结合情况。此外，它们还为两种不同底物浓度下AChE中的底物抑制作用提供了结构上的见解。我们的结构数据表明，中等底物浓度下的底物抑制是由于结合在外周位点的底物分子阻碍了胆碱的排出。在较高浓度下，底物抑制是由于活性位点峡谷内两个底物分子的结合阻止了乙酸盐的排出所致。
