4-a-糖基转移酶的催化区域结构及关键氨基酸解析

Analysis of Structure and Key Amino Acids Existed in 4α Glucanotransferase Catalytic Region

4-a-糖基转移酶(4αGtase)能够作用淀粉产生大环糊精。分别采用精氨酸修饰剂DIC、蛋氨酸修饰剂Ch-T、色氨酸修饰剂NBS、组氨酸修饰剂DEPC、羧基修饰剂EDC、二硫键修饰剂DTT及蛋白变性剂巯基乙醇等对4αGtase进行化学修饰,解析4αGtase催化区域的结构特点和主要氨基酸构成。结果表明,4αGtase活性中心的关键氨基酸为组氨酸、谷氨酸/天冬氨酸,二硫键对于维持4αGtase活性中心的空间结构具有重要作用;该酶可能同时存在淀粉键合区域和催化两个区域,蛋氨酸、色氨酸在两个区域存在状态不尽相同,空间结构的适当变换能够促进该酶与淀粉的结合,从而有利于总酶活的增加。

4α glucanotransferase （4αGtase） has the ability of catalyzing starch to titan large ring cyclodextrin（LR-CI））. In this study,in order to analyze the structure characteristics and key amino acid in the catalytic region,series of amino acids modifiers,such as arginine modifier DIC, methionine modifier Ch-T,tryptophan modifier NBS,histidine modifier DEPC,carboxyl modifier EDC,disulfide boud mndifier DTF and protein denaturant mercaptoethanol,were applied to modify the 4αGtase,respectively. Results showed that Ihe key amino acids existed in active center of 4αtase are histidine,glutamic acid/aspartic acid. The disulfide bond is very important to stabilize the activity of 4αGtase. From these results we deduced that 4αGtase contained two domains inactivity center,methionine and tryptophan are in different region of these two domain. A proper change of spatial structure will promote the combination of enzyme with starch to increase the total activity of 4αGtase.