两种烟碱对映体与胃蛋白酶作用机理的研究
Investigation of Interaction Between Two Enantiomers of Nicotine and Pepsin
作者：杨继(四川大学 化学工程学院，四川 成都 610065;云南中烟工业有限责任公司技术中心，云南 昆明 650231)；杨柳(云南中烟工业有限责任公司技术中心，云南 昆明 650231)；汤建国(云南中烟工业有限责任公司技术中心，云南 昆明 650231)；刘志华(云南中烟工业有限责任公司技术中心，云南 昆明 650231)；陈永宽(云南中烟工业有限责任公司技术中心，云南 昆明 650231)；缪明明(云南中烟工业有限责任公司技术中心，云南 昆明 650231)；唐彬(四川大学 化学工程学院，四川 成都 610065)；李晖(四川大学 化学工程学院，四川 成都 610065)
Author：YANG Ji(School of Chemical Eng., Sichuan Univ., Chengdu 610065, China;Research and Development Center, China Tobacco Yunnan Industry Co. Ltd., Kunming 650231, China)；YANG Liu(Research and Development Center, China Tobacco Yunnan Industry Co. Ltd., Kunming 650231, China)；TANG Jianguo(Research and Development Center, China Tobacco Yunnan Industry Co. Ltd., Kunming 650231, China)；LIU Zhihua(Research and Development Center, China Tobacco Yunnan Industry Co. Ltd., Kunming 650231, China)；CHEN Yongkuan(Research and Development Center, China Tobacco Yunnan Industry Co. Ltd., Kunming 650231, China)；MIAO Mingming(Research and Development Center, China Tobacco Yunnan Industry Co. Ltd., Kunming 650231, China)；TANG Bin(School of Chemical Eng., Sichuan Univ., Chengdu 610065, China)；LI Hui(School of Chemical Eng., Sichuan Univ., Chengdu 610065, China)
收稿日期：2018-09-23 年卷（期）页码：2020,52(1):175-183
期刊名称：工程科学与技术
Journal Name：Advanced Engineering Sciences
关键字：烟碱对映体;胃蛋白酶;作用机理;分子光谱;分子对接
Key words：nicotine enantiomer;pepsin;interaction mechanism;molecular spectroscopy;molecular docking
基金项目：中国烟草总公司科技重大专项项目110201601005（2016xx-05）；云南中烟工业公司科技开发计划项目（2018CP07）；云南中烟科技项目（2019XY02）
中文摘要
利用分子荧光光谱法、紫外可见吸收光谱法结合分子模拟对接技术，重点围绕烟碱（NIC）两种对映体S-NIC和R-NIC对胃蛋白酶（PEP）的荧光猝灭机理、结合常数、结合模式及对酶的活性和二级结构的影响等展开了研究。Stern-Volmer方程拟合结果显示，S-NIC和R-NIC两种对映体与PEP作用时的猝灭常数（Ksv）均随温度的增大而增加，表明二者均通过动态猝灭方式使PEP的内源性荧光强度降低，R-NIC对PEP荧光的猝灭能力明显比S-NIC强，此结果得到紫外可见吸收光谱实验的进一步验证。有效结合常数（Ka）分析可知，R-NIC与PEP的结合明显优于S-NIC。由Arrhenius方程求得S-NIC和R-NIC两种对映体与PEP相互作用的活化能（Ea）分别是12.67 kJ/mol和2.65 kJ/mol，表明R-NIC与PEP碰撞结合更强烈，疏水作用力在此过程中均起主要作用。同步和3维荧光结果表明，NIC两种对映体均诱导PEP的微环境和构象发生部分变化。酶活性研究发现，PEP活性随S-NIC及R-NIC浓度的增加而逐渐降低，相同浓度下R-NIC的抑制比S-NIC更为显著。分子对接结果是：两种对映体在PEP分子中具有相同的结合位点，二者均结合在蛋白分子N端和C端之间的沟槽内。
英文摘要
The fluorescence quenching mechanism, association constant, binding mode and the activity and conformation of pepsin (PEP) between two enantiomers of nicotine (NIC) and PEP were investigated through fluorescence spectroscopy, UV-vis spectroscopy and molecular docking techniques. S-NIC and R-NIC quenched the intrinsic fluorescence of PEP through dynamic quenching mode because of the increase in the Stern-Volmer quenching constant (Ksv) values with the increase in the temperature. The quenching ability of R-NIC was significantly stronger than S-NIC, as evidenced by UV-Vis spectroscopy. The effective association constant (Ka) showed that at the same temperature decreased in the order of R-NIC > S-NIC. The activation energy (Ea) was calculated to be 12.67 kJ/mol and 2.65 kJ/mol for the binding of S-NIC and R-NIC to PEP, respectively. This difference implied an unequal participation of the two enantiomers and R-NIC was more intimate to bind with PEP, and the binding of S-NIC and R-NIC with PEP were both primarily controlled by hydrophobic forces. Synchronous and three-dimensional fluorescence spectroscopy showed that the micro-environmental and conformational of PEP were changed along with the interaction between two enantiomers and PEP. In addition, the activity experiments in vitro indicated that the activity of pepsin was inhibited with increased S-NIC and R-NIC concentration, and the R-NIC was much greater than S-NIC on the activity suppression. Molecular docking study provided the same binding sites, the two enantiomers were located the groove between the N- and C-terminal domains.
上一条：连二硫酸锰碳化干渣热解回收硫酸钠的研究
【关闭】
