棉铃虫可溶型海藻糖酶的化学修饰  

Chemical modification of soluble trehalase from Helicoverpa armigera( Lepidoptera: Noctuiadae)

作　　者：艾东 林荣华[2] 王猛 梁晓贺[1] 于彩虹[1] AI Dong;LIN Rong-Hua;WANG Meng;LIANG Xiao-He;YU Cai-Hong(School of Chemical&Environmental Engineering,China University of Mining&Technology,Beijing 100091,China;Institute for the Control of Agrochemicals,Ministry of Agriculture,Beijing 100125,China;Longkou Entry-exit inspection and Quarantine Bureau,Longkou,Shandong 265700,China)

机构地区：[1]中国矿业大学(北京)化学与环境工程学院,北京100091 [2]农业部农药检定所,北京100125 [3]龙口市出入检验检疫局,山东龙口265700

出　　处：《昆虫学报》2018年第7期801-807,共7页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31171912;30671393)

摘　　要：【目的】本研究旨在通过分析化学修饰剂对棉铃虫Helicoverpa armigera可溶型海藻糖酶活性的影响,以明确海藻糖酶活性中心的结构特点和氨基酸构成。【方法】采用化学修饰方法,测定不同修饰剂处理后棉铃虫5龄幼虫海藻糖酶催化活性的变化,进而通过化学修饰反应失活常数来推测酶活性中心的特定氨基酸残基数量。【结果】采用8 mmol/L水溶性碳二亚胺(carbodiimide,EDC)溶液和25 mmol/L苯甲酰甲醛(phenylglyoxal,PG)溶液分别对棉铃虫5龄幼虫海藻糖酶羧酸基团和精氨酸残基进行修饰后,其活性分别减少81.58%和54.14%,这表明对羧酸基团和精氨酸残基的修饰可有效抑制海藻糖酶活性。底物海藻糖可保护海藻糖酶不受修饰剂的影响。修饰动力学结果显示,海藻糖酶活性中心可能包含1个羧酸基团和2个精氨酸残基。【结论】结果表明,含有羧基的谷氨酸和天冬氨酸是海藻糖酶活性中心的催化残基,精氨酸是维持海藻糖酶活性的必要残基。本研究结果可为开发新型农药提供理论支持。【Aim】This study aims to explore the composition of amino acid residues in the active site of soluble trehalase from Helicoverpa armigera by analyzing the effect of chemical modification on this enzyme activity.【Methods】Chemical modification method was used to investigate the effect of chemical modifier on the soluble trehalase activity in the 5th instar larvae of H.armigera.The number of amino acid residues in active center was obtained from the deactivation rate constant of the modification reaction.【Results】The soluble trehalase activity in the 5th instar larvae of H.armigera was reduced by 81.58%and 54.14%after chemical modification by 8 mmol/L carbodiimide(EDC)and 25 mmol/L phenylglyoxal(PG),respectively,indicating that the modification of carboxylic acid group and arginine residues can inhibit the soluble trehalase activity effectively.The treatment with the substrate trehalose prevented the loss of enzyme activity from the chemical modification.Kinetic studies on chemical modification showed that the active site of soluble trehalase includes one carboxylic acid group and two arginine residues.【Conclusion】These results indicate that two carboxylic amino acids,i.e.,glutamic acid and aspartic acid,are the vital residues of the active site of the soluble trehalase,and arginine is essential for the trehalase activity.These results provide theoretical support for the development of new pesticides.

关 键 词：棉铃虫 海藻糖酶 化学修饰 活性中心 失活常数 

分 类 号：Q966[生物学—昆虫学]