Peng L, Silman I, Sussman J, Goeldner M
Laboratoire de Chimie Bio-organique, URA 1386 CNRS, Faculté de Pharmacie, Université Louis Pasteur Strasbourg, Illkirch, France.
Biochemistry. 1996 Aug 20;35(33):10854-61. doi: 10.1021/bi9529014.
Acetylcholinesterase and butyrylcholinesterase both rapidly hydrolyze the neurotransmitter acetylcholine. The unusual three-dimensional structure of acetylcholinesterase, in which the active site is located at the bottom of a deep and narrow gorge, raises cogent questions concerning traffic of the substrate, acetylcholine, and the products, choline and acetate, to and from the active site. Time-resolved crystallography offers a promising experimental approach to investigate this issue but requires a suitable triggering mechanism to ensure efficient and synchronized initiation of the dynamic process being monitored. Here we characterize the properties of two photolabile triggers which may serve as tools in time-resolved crystallographic studies of the cholinesterases. These compounds are 2-nitrobenzyl derivatives of choline and of carbamylcholine, which generate choline and carbamylcholine, respectively, upon photochemical fragmentation. Both photolabile compounds are reversible inhibitors, which bind at the active sites of acetylcholinesterase and butyrylcholinesterase with inhibition constants in the micromolar range, and both photofragmentation processes occur rapidly and with a high quantum yield, without substantial photochemical damage to the enzymes. Photolysis both of acetylcholinesterase and of butyrylcholinesterase, complexed with a 2-nitrobenzyl derivative of choline, resulted in regeneration of enzymic activity. Photolysis of acetylcholinesterase complexed with the 2-nitrobenzyl derivative of carbamylcholine led to time-dependent inactivation, resulting from carbamylation of acetylcholinesterase, which could be reversed upon dilution, due to decarbamylation. Both sets of experiments demonstrated release of choline within the active site. In the former case, choline was produced photochemically at the active site. In the latter case, choline was generated enzymatically, within the active site, concomitantly with carbamylation of the acetylcholinesterase. The two photolabile compounds may thus serve as complementary probes for time-resolved studies of the route of product release from the active sites of the cholinesterases.
乙酰胆碱酯酶和丁酰胆碱酯酶都能快速水解神经递质乙酰胆碱。乙酰胆碱酯酶不同寻常的三维结构，其活性位点位于一个深而窄的峡谷底部，这就引发了有关底物乙酰胆碱以及产物胆碱和乙酸进出活性位点的运输的令人信服的问题。时间分辨晶体学提供了一种很有前景的实验方法来研究这个问题，但需要一种合适的触发机制来确保对所监测的动态过程进行高效且同步的启动。在此，我们描述了两种光不稳定触发剂的特性，它们可作为胆碱酯酶时间分辨晶体学研究中的工具。这些化合物是胆碱和氨甲酰胆碱的2 - 硝基苄基衍生物，它们在光化学裂解时分别生成胆碱和氨甲酰胆碱。这两种光不稳定化合物都是可逆抑制剂，它们以微摩尔范围内的抑制常数结合在乙酰胆碱酯酶和丁酰胆碱酯酶的活性位点上，并且两种光裂解过程都快速发生且具有高量子产率，对酶没有实质性的光化学损伤。与胆碱的2 - 硝基苄基衍生物复合的乙酰胆碱酯酶和丁酰胆碱酯酶的光解都导致酶活性的再生。与氨甲酰胆碱的2 - 硝基苄基衍生物复合的乙酰胆碱酯酶的光解导致时间依赖性失活，这是由于乙酰胆碱酯酶的氨甲酰化所致，稀释后可因脱氨甲酰化而逆转。两组实验都证明了活性位点内胆碱的释放。在前一种情况下，胆碱是在活性位点光化学产生的。在后一种情况下，胆碱是在活性位点内酶促产生的，并伴随着乙酰胆碱酯酶的氨甲酰化。因此，这两种光不稳定化合物可作为互补探针，用于对胆碱酯酶活性位点产物释放途径的时间分辨研究。
