?-fucosidase-ganglioside interactions. Action of ?-L-fucosidase from the hepatopancreas of Octopus vulgaris on a fucose-containing ganglioside (Fuc-GM1)

A DAniello; A Fiorilli; A Giuliani; B Venerando; G Tettamanti; M Masserini

摘要

palpha-L-Fucosidase, prepared in highly purified form (Mr 70 000-74 000) from Octopus hepatopancreas, was able to hydrolyse a fucose-containing ganglioside, namely Fuc-GM1 (II3NeuAc, IV2Fuc-GgOse4-Cer). The enzyme showed an irregular kinetic behaviour (v/[S] and v/[E] relationships following sigmoidal curves) when working on micellar Fuc-GM1 (Mr of the micelle 500 000), but obeyed regular hyperbolic kinetics when acting on low-Mr substances. It was observed that, on incubation with micellar Fuc-GM1 under the conditions used for the enzyme assay, Octopus alpha-L-fucosidase produced a ganglioside-enzyme complex that was catalytically inactive. This complex had an Mr exceeding 500 000 and a ganglioside/protein ratio of 4:1 (w/w), which is consistent with a stoichiometric combination of one ganglioside micelle with two enzyme molecules. Inactivation of alpha-L-fucosidase by formation of the corresponding complexes was also obtained with micellar gangliosides GM1 (II3NeuAc-GgOse4-Cer), GD1a (II3NeuAc, IV3NeuAc-GgOse4-Cer) and GT1b [II3(NeuAc)2,IV3-NeuAc-GgOse4-Cer], which are not substrates for the enzyme, indicating that the ganglioside micelles per se act as enzyme inhibitors. However, alpha-L-fucosidase easily forms a Fuc-GM1-alpha-L-fucosidase complex, displaying regular Michaelis-Menten kinetics. Therefore the anomalous behaviour exhibited by alpha-L-fucosidase on micellar Fuc-GM1 is likely due to formation of the complex, which separates the fucosyl linkage from the active site of the complexed enzyme, but makes it available to the enzyme in the free form./p

机译：由章鱼肝胰腺以高度纯化的形式（Mr 70 000-74 000）制备的α-L-岩藻糖苷酶能够水解含岩藻糖的神经节苷脂，即Fuc-GM1（II3NeuAc，IV2Fuc-GgOse4-Cer）。在胶束Fuc-GM1（胶束的Mr 500 000）上工作时，该酶表现出不规则的动力学行为（遵循S形曲线的v / [S]和v / [E]关系），但在低分子量的Fuc-GM1上则服从规则的双曲线动力学。先生物质。观察到，在用于酶测定的条件下与胶束Fuc-GM1一起孵育时，章鱼α-L-岩藻糖苷酶产生了一种神经节苷脂-酶复合物，该复合物是催化失活的。该复合物的Mr超过500 000，神经节苷脂/蛋白质之比为4：1（w / w），这与一个神经节苷脂胶束与两个酶分子的化学计量结合是一致的。还通过胶束神经节苷脂GM1（II3NeuAc-GgOse4-Cer），GD1a（II3NeuAc，IV3NeuAc-GgOse4-Cer）和GT1b [II3（NeuAc）2，IV3-NeuAc）的形成，通过形成相应的复合物而使α-L-岩藻糖苷酶失活。 -GgOse4-Cer]，它们不是酶的底物，表明神经节苷脂胶束本身起酶抑制剂的作用。但是，α-L-岩藻糖苷酶很容易形成Fuc-GM1-α-L-岩藻糖苷酶复合物，表现出规则的Michaelis-Menten动力学。因此，α-L-岩藻糖苷酶在胶束Fuc-GM1上表现出的异常行为可能是由于复合物的形成，该复合物将岩藻糖基键与复合酶的活性位点分开，但使其以游离形式可用于该酶。

?-fucosidase-ganglioside interactions. Action of ?-L-fucosidase from the hepatopancreas of Octopus vulgaris on a fucose-containing ganglioside (Fuc-GM1)

摘要

著录项

相关主题

期刊订阅