Ito K, Yamamoto T, Minamiura N
Faculty of Science, Osaka City University.
J Biochem. 1987 Aug;102(2):359-67. doi: 10.1093/oxfordjournals.jbchem.a122062.
Phosphodiesterase I [EC 3.1.4.1] was purified from normal human urine in a highly purified state free from phosphodiesterase II, RNase, DNase I, DNase II, and phosphatase by column chromatographies of DEAE-Toyopearl, butyl-Toyopearl, Affi-Gel blue, and Sephadex G-150. The molecular weight of the enzyme was 1.9 x 10(5) and the pH optimum around 9.0 with p-nitrophenyl deoxythymidine 5'-phosphate as the substrate. The enzyme hydrolyzed the 3'-5' linkage of various dinucleoside monophosphates at approximately the same rate and the phosphodiester bonds of cyclic 3',5'-mononucleotides to produce mononucleoside 5'-phosphate. The enzyme also hydrolyzed ADP to 5'-AMP and Pi, ATP to 5'-AMP and PPi, and NAD+ to 5'-AMP and NMN. The enzyme activity was abolished by removal of metal ions with EDTA, and the metal-free enzyme was reactivated on the addition of Zn2+. The enzyme activity was also abolished by some reducing agents and the inhibition was reversed by Zn2+. The metal-free enzyme was less stable than the native enzyme, and Zn2+ and Co2+ restored the stability of the metal-free enzyme to the level of the native enzyme. The enzyme degraded oligonucleotides and high molecular nucleotides stepwise from the 3'-termini to give 5'-mononucleotides. The enzyme hydrolyzed single-stranded DNA more preferentially than double-stranded DNA. The enzyme also nicked superhelical covalently closed circular phi X174 DNA to yield first open circular DNA and then linear DNA.
磷酸二酯酶I [EC 3.1.4.1]通过DEAE-琼脂糖凝胶、丁基-琼脂糖凝胶、Affi-Gel蓝和葡聚糖凝胶G-150柱色谱法从正常人尿液中以高度纯化的状态分离得到，该状态不含磷酸二酯酶II、核糖核酸酶、脱氧核糖核酸酶I、脱氧核糖核酸酶II和磷酸酶。该酶的分子量为1.9×10⁵，以对硝基苯基脱氧胸苷5'-磷酸为底物时，最适pH约为9.0。该酶以大致相同的速率水解各种二核苷单磷酸的3'-5'键以及环3',5'-单核苷酸的磷酸二酯键，生成5'-单磷酸核苷。该酶还将ADP水解为5'-AMP和Pi，将ATP水解为5'-AMP和PPi，将NAD⁺水解为5'-AMP和NMN。用EDTA去除金属离子会使酶活性丧失，添加Zn²⁺后无金属酶会重新激活。一些还原剂也会使酶活性丧失，Zn²⁺可逆转这种抑制作用。无金属酶比天然酶稳定性差，Zn²⁺和Co²⁺可将无金属酶的稳定性恢复到天然酶的水平。该酶从3'-末端逐步降解寡核苷酸和高分子量核苷酸，生成5'-单核苷酸。该酶对单链DNA的水解优先于双链DNA。该酶还能切割超螺旋共价闭合环状φX174 DNA，首先产生开环DNA，然后产生线性DNA。
