维生素K_(3)激活黄嘌呤氧化酶的动力学和分子机制

Kinetics and molecular mechanism of vitamin K_(3)as xanthine oxidase activator

目的考察维生素K_(3)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16μmol·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(K_(m))。黄嘌呤在K_(m)浓度下,三点法(维生素K_(3)1,10和100μmol·L^(-1))检测维生素K_(3)激活剂的活性,多点法(维生素K_(3)1,2,5,10,20,50,100,200和400μmol·L^(-1))测定其激活XO的半数有效浓度(EC_(50))。使用1/2EC_(50),EC_(50)和2EC_(50)维生素K_(3)进行动力学参数(K_(m)和V_(max))的测定和双倒数曲线的拟合,考察不同浓度维生素K_(3)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K_(3)之间的互作机制。结果XO介导黄嘌呤氧化反应的K_(m)为4.71μmol·L^(-1)。作为该反应的激活剂,维生素K_(3)浓度依赖性激活XO(logistic拟合公式y=A2+(A1-A2)/1+(x/x_(0))^p),且EC_(50)为32.0μmol·L^(-1)。反应的动力学参数在加入维生素K_(3)后发生变化,K_(m)值随维生素K_(3)浓度增加而减小(4.71~1.34μmol·L^(-1)),而V_(max)值随维生素K_(3)浓度增加而增加(0.08~1.31μmol·min^(-1)·g^(-1)),最终导致V_(max)/K_(m)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K_(3)对XO的激活类型为混合型。分子对接结果显示,维生素K_(3)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K_(3)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 K_(3)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 16μmol·L^(-1)at 37℃for 90 min.The Michaelis constant(K_(m))of the reaction of xanthine oxidation was determined using the liquid chromatography diode array method.At the concentration of K_(m),the three-point method(1,10 and 100μmol·L^(-1))was used to detect the activity of vitamin K_(3)activators.The multi-point method(vitamin K_(3)1,2,5,10,20,50,100,200 and 400μmol·L^(-1))was adopted to determine the half effective concentration(EC_(50))of activated XO.Kinetic parameters(K_(m)and V_(max))and the fit of double reciprocal curves were determined via vitamin K_(3)of 1/2EC_(50),EC_(50)and 2EC_(50).The changes in kinetic behavior at different concentrations of vitamin K_(3)were observed and their types of activation were analyzed.The interactions between XO and activator vitamin K_(3)were explored via molecular docking.RESULTS The K_(m)of XO-mediated xanthine oxidation reac⁃tion was 4.71μmol·L^(-1).As an activator of this reaction,vitamin K_(3)activated XO in a concentration-dependent manner(according to the logistic fitting formula y=A2+(A1-A2)/(1+(x/x0)^p),with an EC_(50)of 32.0μmol·L^(-1).The kinetic parameters also changed after the addition of vitamin K_(3).The K_(m)value decreased(4.71-1.34μmol·L^(-1))with the increase of vitamin K_(3)concentrations,while the V_(max)value increased(0.08-1.31μmol·min^(-1)·g^(-1)),leading to an increase in V_(max)/K_(m)(17.0-977.6 mL·min·g^(-1)).In addition,the double reciprocal curve fitting found that the activation type of vitamin K_(3)on XO was mixed.The molecular docking results showed that vitamin K_(3)bound to the molybdopterin domain of XO and maintained hydrogen bonding interactions with Arg599 and Ser605.CONCLUSION Vitamin K_(3)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.