中国药理学与毒理学杂志2024，Vol.38Issue(2)：113-119,7.DOI:10.3867/j.issn.1000-3002.2024.02.005
维生素K3激活黄嘌呤氧化酶的动力学和分子机制
Kinetics and molecular mechanism of vitamin K3 as xanthine oxidase activator
摘要
Abstract
OBJECTIVE To investigate the activation of xanthine oxidase(XO)from the human liver by vitamin K3 and the mechanism.METHODS Using human liver S9(0.1 g·L-1)as the source,XO was incubated with substrate xanthine of 0,2,4,8,and 16 μmol·L-1 at 37℃ for 90 min.The Michaelis constant(Km)of the reaction of xanthine oxidation was determined using the liquid chromatography diode array method.At the concentration of Km,the three-point method(1,10 and 100 μmol·L-1)was used to detect the activity of vitamin K3 activators.The multi-point method(vitamin K3 1,2,5,10,20,50,100,200 and 400 μmol·L-1)was adopted to determine the half effective concentration(EC50)of activated XO.Kinetic parameters(Km and Vmax)and the fit of double reciprocal curves were determined via vitamin K3 of 1/2EC50,EC50 and 2EC50.The changes in kinetic behavior at different concentrations of vitamin K3 were observed and their types of activation were analyzed.The interactions between XO and activator vitamin K3 were explored via molecular docking.RESULTS The Km of XO-mediated xanthine oxidation reac-tion was 4.71 μmol·L-1.As an activator of this reaction,vitamin K3 activated XO in a concentration-dependent manner(according to the logistic fitting formula y=A2+(A1-A2)/(1+(x/x0)^p),with an EC50 of 32.0 μmol·L-1.The kinetic parameters also changed after the addition of vitamin K3.The Km value decreased(4.71-1.34 μmol·L-1)with the increase of vitamin K3 concentrations,while the Vmax value increased(0.08-1.31 μmol·min-1·g-1),leading to an increase in Vmax/Km(17.0-977.6 mL·min·g-1).In addition,the double reciprocal curve fitting found that the activation type of vitamin K3 on XO was mixed.The molecular docking results showed that vitamin K3 bound to the molybdopterin domain of XO and maintained hydrogen bonding interactions with Arg599 and Ser605.CONCLUSION Vitamin K3 is an activator of XO,which can form hydrogen bonds with Arg599 and Ser605 in the XO domain,regu-late its affinity with the substrate xanthine,activate XO and increase the uric acid level.关键词
维生素K3/黄嘌呤氧化酶/变构调节/分子对接Key words
vitamin K3/xanthine oxidase/allosteric regulation/molecular docking分类
医药卫生引用本文复制引用
刘礼,赵文静,肖丽君,齐晓怡,吕沐瀚,梁思成,吴敬敬..维生素K3激活黄嘌呤氧化酶的动力学和分子机制[J].中国药理学与毒理学杂志,2024,38(2):113-119,7.基金项目
国家自然科学基金(81672458) （81672458）
国家自然科学基金(82003850) （82003850）
四川省科技规划项目(2021JDTD0003) （2021JDTD0003）
四川省科技规划项目(2022NSFSC0576) （2022NSFSC0576）
四川省科技计划(2022YFS0626) （2022YFS0626）
四川省科技计划(2022YFS0631) National Natural Science Foundation of China(81672458) （2022YFS0631）
National Natural Science Foundation of China(82003850) （82003850）
Sichuan Provincial Science and Technology Planning Project(2021JDTD0003) （2021JDTD0003）
Sichuan Provincial Science and Technology Planning Project(2022NSFSC0576) （2022NSFSC0576）
Sichuan Provincial Science and Technology Plan(2022YFS0626) （2022YFS0626）
and Sichuan Provincial Science and Technology Plan(2022YFS0631) （2022YFS0631）
