Mg^2+介导底物亲和力的改变增加黍子过氧化物酶的磷酸酶活性  

The Affinity Change of Mg^2+-mediated Substrates Increases the Phosphatase Activity of Proso Millet Peroxidase

作　　者：崔晓东 王柯 王转花 CUI Xiao-Dong;WANG Ke;WANG Zhuan-Hua(Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education,Institute of Biotechnology,Taiyuan 030006,China;School of Life Sciences,Shanxi University,Taiyuan 030006,China)

机构地区：[1]化学生物学与分子工程教育部重点实验室,山西大学生物技术研究所,太原030006 [2]山西大学生命科学学院生物工程系,太原030006

出　　处：《中国生物化学与分子生物学报》2020年第3期310-318,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.31300653);山西省应用基础研究计划项目(No.201801D121192);山西省高等学校科技创新项目(No.201802020)资助。

摘　　要：黍子过氧化物酶(proso millet peroxidase,PmPOD)具有磷酸酶活性,可以断裂DNA中磷酸二酯键及脱氧核糖核苷酸(dNMPs)中磷酸单酯键。在此反应过程中,Mg^2+显著增强PmPOD的磷酸酶活性,但其具体的机制尚不明确。本文采用紫外-可见分光光谱法和荧光光谱法,研究了以dNMPs为底物时,Mg^2+对PmPOD磷酸酶活性的影响,并对其反应机制进行了初步的探究。紫外-可见分光光度法结果表明:Mg^2+介导了PmPOD与底物的相互作用,但Mg^2+并未直接与PmPOD发生相互作用。荧光光谱进一步表明,在Mg^2+存在的情况下,dNMPs对PmPOD内源荧光淬灭方式发生变化,由动态淬灭转变为静态淬灭。同时还发现,dNMPs与PmPOD的结合常数Ka增加约2~10倍(与不存在Mg^2+条件相比),依次为:KadCMP>KadGMP>KadTMP>KadAMP。高效液相色谱表明,Mg^2+可增强PmPOD水解dNMPs的速率3~13倍,且水解速率VdCMP>VdGMP>VdTMP>VdAMP,与结合常数的变化一致。因此,我们得出结论,PmPOD发挥磷酸酶活性时,Mg^2+首先与dNMPs形成中间产物,这一中间产物更适合与PmPOD形成复合物,增大了底物dNMPs与PmPOD结合常数,进而加速了PmPOD水解dNMPs。本研究为Mg^2+在过氧化物酶催化DNA水解的机制提供了相关依据,为研究金属离子增强蛋白酶活性的机制提供了理论基础。Proso millet peroxidase(PmPOD)has phosphatase activity,which can break the phosphate diester bond in DNA and the phosphate monoester bond in deoxyribonucleotides(dNMPs).In this process,Mg^2+can greatly enhance the phosphatase activity of PmPOD,but the specific mechanism is still unclear.In this paper,ultraviolet-visible(UV-vis)and fluorescence spectroscopy were used to further study the effect of Mg^2+on the phosphatase activity of PmPOD with dNMPs as the substrates,and the mechanism of its combination reaction was also explored.UV-vis spectroscopy showed that Mg^2+mediates the interaction between PmPOD and substrates,but Mg^2+may not directly interact with PmPOD.Fluorescence spectroscopy further showed that in the presence of Mg^2+,dNMPs changed the mechanism of intrinsic fluorescence quenching of PmPOD,from dynamic quenching to static quenching.At the same time,we also found that the binding constant Ka of dNMPs and PmPOD increased by about 2-10 times in the presence of Mg^2+(compared with the absence of Mg^2+condition),KadCMP>KadGMP>KadTMP>KadAMP.High performance liquid chromatography showed that Mg^2+could improve the hydrolysis rate of PmPOD by 3-13 times with dNMPs as the substrate,and the hydrolysis rate were VdCMP>VdGMP>VdTMP>VdAMP,which was consistent with the changes of binding constant of dNMPs.Therefore,we conclude that Mg^2+increases the binding constant of dNMPs and PmPOD through forming suitable substrates for PmPOD,thus accelerating the hydrolysis of dNMPs by PmPOD.This study provides the relevant basis for the mechanism of Mg^2+in the peroxidase catalyzed DNA hydrolysis,and provides the theoretical basis for studying the mechanism of metal ions enhancing the protease activity.

关 键 词：黍子过氧化物酶 磷酸酶 镁离子 荧光光谱 脱氧核糖核苷-5'-一磷酸 

分 类 号：Q516[生物学—生物化学]