达托霉素作为新的胰蛋白酶激活剂的初探

doi:10.13523/j.cb.20171004

中国生物工程杂志

2017, Vol. 37

Issue (10): 26-32 DOI: 10.13523/j.cb.20171004

研究报告

达托霉素作为新的胰蛋白酶激活剂的初探

杨恬然, 吴世杰, 辛明秀

北京师范大学生命科学学院基因工程药物与生物技术北京市重点实验室北京 100875

Study on Daptomycin as a New Trypsin Activator

YANG Tian-ran, WU Shi-jie, XIN Ming-xiu

College of Life Sciences, Beijing Normal University, Beijing Key Laboratory of Genetically Engineered Drug and Biotechnology, Beijing 100875, China

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摘要： 首次发现达托霉素是一种新的胰蛋白酶激活剂，当胰蛋白酶与达托霉素的物质的量的比在反应时达到34.05时，达托霉素对胰蛋白酶比活力的激活率平均达到32.92%。通过计算机模拟技术对达托霉素与胰蛋白酶以及达托霉素对胰蛋白酶底物复合物分别进行分子对接，并利用等温滴定量热法（Isothermal titration calorimetry，ITC）验证模拟结果。研究发现达托霉素与胰蛋白酶的活性中心位置很靠近，达托霉素链状中的天冬氨酸的R基与酶活性中心的组氨酸-57发生了氢键相互作用，达托霉素使酶和底物复合物结构更加稳定，从而有利于催化作用。ITC结果表明胰蛋白酶上具有1个达托霉素结合位点，解离常数Kd为17.83 μM，摩尔结合焓△H为237.9±28.17 cal/mol，摩尔结合熵△S为22.5 cal/mol·deg，这些结果从热力学角度支持了分子对接结果。研究发现达托霉素除具有抗革兰氏阳性耐药性细菌的功能外，还能促进胰蛋白酶比活这一新的功能，是一种新的胰蛋白酶激活剂。

关键词： 达托霉素; 胰蛋白酶; 分子对接; 酶激活剂

Abstract: The activity of trypsin was increased 32.92% on average when the amount of substance rate between trypsin and daptomycin is 34.05, which indicated that daptomycin is a new trypsin activator. Daptomycin-trypsin and daptomycin-trypsin-substrate complex were studied by the molecular docking, and isothermal titration calorimetry (ITC) was used to support the result of molecular docking. It showed that daptomycin was close to the activity center of trypsin, and the R group of daptomycin was interacted with histidine-57 of trypsin, and this histidine-57 located in the activity center of the enzyme. The complex structure between enzyme and substrate is more stable when the daptomycin is presence, which is beneficial to the catalytic effect. The ITC results demonstrated that a daptomycin binding site was located in trypsin, and the dissociation constant Kd is 17.83 μM, molar combustion enthalpy △H is 237.9±28.17 cal/mol, molar binding entropy △S is 22.5 cal/mol/deg, and all the results support the molecular docking results. It indicated that daptomycin is a new trypsin activator and it will have application in proteomics and other fields.

Key words: Daptomycin Trypsin Enzyme activator Molecular docking

收稿日期: 2017-04-13 出版日期: 2017-10-25

ZTFLH:

Q819

基金资助: 国家自然科学基金资助项目（31470251）

通讯作者: 辛明秀,xinmingxiu@bnu.edu.cn E-mail: xinmingxiu@bnu.edu.cn

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引用本文:

杨恬然, 吴世杰, 辛明秀. 达托霉素作为新的胰蛋白酶激活剂的初探[J]. 中国生物工程杂志, 2017, 37(10): 26-32.

YANG Tian-ran, WU Shi-jie, XIN Ming-xiu. Study on Daptomycin as a New Trypsin Activator. China Biotechnology, 2017, 37(10): 26-32.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171004 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I10/26

[1]	Kerry L T, Pharm D, Michael J R, et al. Daptomysin. Pharmacotherapy,2004,24(1):41-57.
[2]	Andres B F, Ferdinand V, Ralf S, et al. Daptomycin:a novel lipopeptide antibiotic against Gram-positive pathogens. Infect Drug Resist,2010,3:95-101.
[3]	Robert S, Markus R, Wolfgang G, et al. Daptomycin:a review 4 years after first approval. Pharmacology,2008,81(2):79-91.
[4]	Elien V A, Michael M, Lennart M. Getting intimate with trypsin,the leading protease in proteomics. Mass Spectrom Rev,2013, 32(6):453-465.
[5]	Marc R W, Keith L W, Ron D A, et al. Proteome Research:New Frontiers in Functional Genomics. Berlin:Springer Berlin Heidelberg,1997.1-12.
[6]	Mathias W, Judith S, Hannes H, et al. Mass-spectrometry-based draft of the human proteome. Nature,2014,509(7502):582-587.
[7]	Michael B S, Nathan C V, David L T, et al. Characterization of the 70S ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach. J Proteome Res, 2004,3(5):965-978.
[8]	王胜.蛋白质组学高效酶解新技术.上海:复旦大学,2008,38-39. Wang S. A series of new strategies of high efficient proteolysis for proteomic analysis. Doctor Dissertation. Shanghai:Fudan University, 2008,38-39.
[9]	江锟.鲈鱼蛋白的酶解工艺优化及活性肽的功能特性研究.湖北:华中农业大学,2013,5-6. Jiang K. Studies of bass protein hydrolysis process optimization and active peptide functional propertie. Master Dissertation. Hubei:Huazhong Agricultural University, 2013,5-6.
[10]	刘宝阳.蛋白质组学技术的改进及对肝细胞肝癌的研究.天津:天津医科大学,2014,5-10. Liu B Y. Proteomics technology improvement and the study of HCC. Master Dissertation. Tianjin:Tianjin Medical University,2014,5-10.
[11]	王镜岩,朱圣庚, 徐长法. 生物化学教程. 北京:高等教育出版社, 2008,161-163. Wang J Y, Zhu S G, Xu C F. Essential Biochemistry. Beijing:Higher Education Press, 2008.161-163.
[12]	林勇,周健,毕登,等.脱氧胆酸钠(SDC)促进的蛋白质酶解与鉴定.中国科技论文在线精品论文. 2009,2(7):1-6. Lin Y, Zhou J,Bi D,et al. Sodium deoxycholate-improved enzymolysis and identification of proteins. The Best Online Papers in China Scientific and Technological Papers, 2009, 2(7):1-6.
[13]	戴超. 脂肽类生物表面活性剂的提取工艺及其乳化性的研究.南京:南京农业大学,2013,10-11. Dai C. Lipopeptide biosurfactant process and its emulsifying properties. Doctor Dissertation.Nanjing:Nanjing Agricultural University, 2013,10-11.
[14]	Anson M L. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J Gen Physiol, 1938,22(1):79-89.
[15]	Carrie C E. Sigma's non-specific protease activity assay -casein as a substrate. J Vis Exp, 2008,19(19):899.
[16]	Hongtao Zhao. Tutorial for LeDock. Switzerland:Lephar Research,2015.3-5.
[17]	张幼怡.蛋白质-蛋白质相互作用方法与应用.北京:北京大学医学出版社,2008.39-57. Zhang Y Y. Protein-Protein Interactions Methods and Applications. Beijing:Peking University Medical Press,2008.39-57.
[18]	Emesto F, Obdulio L M, Martin S. Isothermal titration. Analytical Chemistry,1990,62(18):950-959.
[19]	朱少娟,施用晖,乐国伟.超声波对胰蛋白酶水解酪蛋白的影响.食品与生物技术学报, 2005,24(2):50-54. Zhu S J, Shi Y H, Le G W. Effects of ultrasound on the hydrolysis of casein by trypsin. Journal of Food Science and Biotechnology,2005, 24(2):50-54.
[20]	胡毅.三疣梭子蟹消化酶性质的研究.青岛:中国海洋大学,2003,37-44. Hu Y. Studies on characterization of the digestive enzymes from Portunus. Master Dissertation. Qingdao:Graduate School of Ocean University of China, 2003,37-44.
[21]	宋翼行,黎春怡,黄卓烈,等.甲醇对胰蛋白酶催化活性影响的机理研究.生命科学研究,2010,14(4):311-315. Song Y H, Li C Y, Huang Z L, et al. Study on the effects of carbinol solvents on trypsin activity. Life Science Research,2010, 14(4):311-315.
[22]	李黎,谭进,刘志芳.曲酸与胰蛋白酶相互作用的光谱学研究.四川大学学报(自然科学版),2009,46(5):1466-1472. Li L, Tan J, Liu Z F, et al. Studies on the interaction between kojic acid and trypsin by fluorescence spectrophotometry. Journal of Sichuan University (Natural Science Edition),2009, 46(5):1466-1472.
[23]	Robert D A, Dennis M, Francis P T, et al. The safety and efficacy of daptomycin for the treatment of complicated skin and skin-structure infections. Clinical Infectious Diseases,2004,38(12):1673-1681.
[24]	Fowler V G, Boucher H W, Corey G R. Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphylococcus aureus. N Engl J Med,2006,355(7):653-665.
[25]	Lamp K C, Friedrich L V, Mendez-Vigo L, et al. Clinical experience with daptomycin for the treatment of patients with osteomyelitis. Am J Med,2007,120(10):13-20.
[26]	Jared A S, Lawrence I M, Andrew D G, et al. Inhibition of daptomycin by pulmonary surfactant:in vitro modeling and clinical impact. J Infect Dis, 2005,191(12):2149-2152.
[27]	Zhang T H. Daptomycin:mechanism of action and bacterial resistance. Canada:The University of Waterloo,2015,5-34.
[28]	Rolston K V, McConnell S A, Brown J, et al. Daptomycin use in patients with cancer and neutropenia:data from a retrospective registry. Adv Hematol,2010,8(4):249-256, 290.

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