Smialowski-Fléter S, Moulin A, Villard C, Puigserver A
Laboratoire de Biochimie et Biologie de la Nutrition, Faculté des Sciences et Techniques de St-Jérôme, Marseille, France.
Eur J Biochem. 2000 Apr;267(8):2227-34. doi: 10.1046/j.1432-1327.2000.01224.x.
CNBr fragments from porcine intestinal glycerol-ester hydrolase were separated by SDS/PAGE under reducing and nonreducing conditions, and their amino-acid sequences were analysed. Two intra-chain disulfide bridges were identified, namely Cys70-Cys99 (loop A) and Cys256-Cys267 (loop B). As the Cys71 sulfhydryl group could not be alkylated with iodoacetamide, it is suggested that the residue is blocked rather than being present in the free form. The two disulfide bridges of intestinal glycerol-ester hydrolase are present in the cholinesterase family, although the enzyme showed only about 35% identity with these proteins. Furthermore, the finding that glycerol-ester hydrolase was partly inactivated under reducing conditions suggests that one or both disulfide bridges are important for the enzyme conformation. Lastly, glycerol-ester hydrolase was also found to hydrolyse cholinergic substrates, although residues Trp86 and Asp74 which are considered to be the main constituents of the 'anionic' subsite responsible for substrate binding in cholinesterases were absent from loop A. Other amino-acid residues in the glycerol-ester hydrolase may therefore be responsible for the binding of cholinergic substrates to the enzyme.
在还原和非还原条件下，通过SDS/PAGE对猪肠道甘油酯水解酶的溴化氰片段进行分离，并分析其氨基酸序列。鉴定出两条链内二硫键，即Cys70-Cys99（环A）和Cys256-Cys267（环B）。由于Cys71巯基不能被碘乙酰胺烷基化，表明该残基被封闭而非以游离形式存在。肠道甘油酯水解酶的两条二硫键存在于胆碱酯酶家族中，尽管该酶与这些蛋白质的同一性仅约为35%。此外，甘油酯水解酶在还原条件下部分失活的发现表明，一条或两条二硫键对酶的构象很重要。最后，还发现甘油酯水解酶可水解胆碱能底物，尽管环A中不存在被认为是胆碱酯酶中负责底物结合的“阴离子”亚位点主要成分的Trp86和Asp74残基。因此，甘油酯水解酶中的其他氨基酸残基可能负责胆碱能底物与该酶的结合。
