Amici A, Ciccioli K, Naponelli V, Raffaelli N, Magni G
Istituto di Biotecnologie Biochimiche, Università Politecnica Delle Marche, Via Ranieri 67, 60131, Ancona, Italy.
Cell Mol Life Sci. 2005 Jul;62(14):1613-20. doi: 10.1007/s00018-005-5135-y.
Human erythrocyte pyrimidine 5'-nucleotidase, PN-I, catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates. The enzyme also possesses phosphotransferase activity, transferring phosphate groups between pyrimidine nucleoside monophosphates and various pyrimidine nucleosides. Deficiency of the enzyme activity is associated with a hemolytic anemia. PN-I cDNA has been expressed in Escherichia coli, yielding a fully active recombinant enzyme, which was purified to homogeneity and extensively characterized. Multiple sequence alignment of PN-I and homologues proteins revealed the existence of conserved regions, whose importance in catalysis was examined by performing experiments designed to intercept covalent intermediates as strongly suggested by our previous kinetic studies. Furthermore, a functional analysis of the enzyme was carried out through site-directed mutagenesis designed on the basis of the sequence of the identified conserved regions as well as mutations observed in PN-I-deficient patients.
人红细胞嘧啶5'-核苷酸酶PN-I催化嘧啶核苷单磷酸的去磷酸化反应。该酶还具有磷酸转移酶活性，可在嘧啶核苷单磷酸和各种嘧啶核苷之间转移磷酸基团。酶活性缺乏与溶血性贫血相关。PN-I的cDNA已在大肠杆菌中表达，产生了一种具有完全活性的重组酶，该重组酶被纯化至同质，并进行了广泛的特性鉴定。PN-I与同源蛋白的多序列比对揭示了保守区域的存在，根据我们之前的动力学研究强烈提示，通过设计旨在截留共价中间体的实验来检验其在催化中的重要性。此外，基于鉴定出的保守区域序列以及在PN-I缺乏患者中观察到的突变，通过定点诱变对该酶进行了功能分析。
