维生素K3激活黄嘌呤氧化酶的动力学和分子机制OACSTPCD
Kinetics and molecular mechanism of vitamin K3 as xanthine oxidase activator
目的 考察维生素K3对人肝来源的黄嘌呤氧化酶(XO)的激活作用及其作用机制.方法 以人肝S9(0.1 g·L-1)作为XO来源,分别与底物黄嘌呤(0,2,4,8和16 μmol·L-1)在37℃孵育90 min,液相色谱二极管阵列法测定反应的米氏常数(Km).黄嘌呤在Km浓度下,三点法(维生素K3 1,10和100 μmol·L-1)检测维生素K3激活剂的活性,多点法(维生素K3 1,2,5,10,20,50,100,200和400 μmol·L-1)测定其激活XO的半数有效浓度(EC50).使用1/2EC50,EC50和2EC50维生素K3进行动力学参数(Km和Vmax)的测定和双倒数曲线的拟合,考察不同浓度维生素K3存在下的动力学行为变化,并分析其激活类型.最后,通过分子对接技术探究XO与维生素K3之间的互作机制.结果 XO介导黄嘌呤氧化反应的Km为4.71 μmol·L-1.作为该反应的激活剂,维生素K3 浓度依赖性激活XO(logistic拟合公式y=A2+(A1-A2)/1+(x/x0)^p),且EC50 为32.0 μmol·L-1.反应的动力学参数在加入维生素K3后发生变化,Km值随维生素K3浓度增加而减小(4.71～1.34 μmol·L-1),而Vmax值随维生素K3浓度增加而增加(0.08～1.31 μmol·min-1·g-1),最终导致Vmax/Km增加(17.0～977.6 mL·min-1·g-1).此外,双倒数曲线拟合表明维生素K3对XO的激活类型为混合型.分子对接结果显示,维生素K3结合于XO的钼喋呤结构域,与Arg599和Ser605形成氢键相互作用.结论 维生素K3是XO的激活剂,可与XO结构域中的Arg599和Ser605形成氢键,调节其与底物黄嘌呤的亲和力,达到激活XO而升尿酸的效果.
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.
刘礼;赵文静;肖丽君;齐晓怡;吕沐瀚;梁思成;吴敬敬
西南医科大学附属医院消化内科,四川 泸州 646000西南医科大学附属医院皮肤科,四川 泸州 646000大连医科大学药学院临床药理学系,辽宁 大连 116044
药学
维生素K3黄嘌呤氧化酶变构调节分子对接
vitamin K3xanthine oxidaseallosteric regulationmolecular docking
《中国药理学与毒理学杂志》 2024 (002)
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国家自然科学基金(81672458);国家自然科学基金(82003850);四川省科技规划项目(2021JDTD0003);四川省科技规划项目(2022NSFSC0576);四川省科技计划(2022YFS0626);四川省科技计划(2022YFS0631) National Natural Science Foundation of China(81672458);National Natural Science Foundation of China(82003850);Sichuan Provincial Science and Technology Planning Project(2021JDTD0003);Sichuan Provincial Science and Technology Planning Project(2022NSFSC0576);Sichuan Provincial Science and Technology Plan(2022YFS0626);and Sichuan Provincial Science and Technology Plan(2022YFS0631)
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