Durán R, Cerveñansky C, Dajas F, Tipton K F
Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay.
Biochim Biophys Acta. 1994 Dec 15;1201(3):381-8. doi: 10.1016/0304-4165(94)90066-3.
Fasciculin 2 (FAS) is a 61 amino acid peptide present in Dendroaspis angusticeps snake venom, with a selective and potent inhibitory activity towards acetylcholinesterase (AChE). The specific interaction of FAS with peripheral sites present in Electrophorus electricus AChE (Ki = 0.04 nM FAS) was investigated by chemical modification with N,N-dimethyl-2-phenylaziridinium (DPA) in the presence of active or peripheral anionic site protective agents. An enzyme was obtained that compared to the native AChE is 10(6)-times less sensitive to FAS, is fully inhibited by edrophonium and tacrine, and is 25-170-times less sensitive to several peripheral site ligands. Characterization of catalytic functions showed that Km for acetylthiocholine was 4-fold lower in the DPA-modified enzyme, whereas Km for phenylacetate remained the same. Values for Kcat determined with both substrates were unchanged. Diminished catalytic efficiency reflects that hydrolysis and/or supply of cationic substrates to the active site was affected by DPA reaction at a peripheral site. Previous data implicate Trp-279 (Torpedo AChE sequence numbering) as the residue actually involved in DPA modification. Our results strongly support FAS binding to an AChE peripheral site which partially overlaps the site of other peripheral site ligands including acetylthiocholine.
束丝菌素2（FAS）是一种存在于绿曼巴蛇毒液中的由61个氨基酸组成的肽，对乙酰胆碱酯酶（AChE）具有选择性且强效的抑制活性。在活性或外周阴离子位点保护剂存在的情况下，通过用N，N - 二甲基 - 2 - 苯基氮丙啶（DPA）进行化学修饰，研究了FAS与电鳗AChE中存在的外周位点的特异性相互作用。获得了一种酶，与天然AChE相比，它对FAS的敏感性降低了10^6倍，被依酚氯铵和他克林完全抑制，并且对几种外周位点配体的敏感性降低了25 - 170倍。催化功能的表征表明，DPA修饰的酶中乙酰硫代胆碱的Km降低了4倍，而苯乙酸的Km保持不变。用两种底物测定的Kcat值未改变。催化效率的降低反映出阳离子底物向活性位点的水解和/或供应受到外周位点DPA反应的影响。先前的数据表明Trp - 279（电鳐AChE序列号编号）是实际参与DPA修饰的残基。我们的结果有力地支持了FAS与AChE外周位点的结合，该位点部分重叠于包括乙酰硫代胆碱在内的其他外周位点配体的位点。
