Phosphorylation of cytidine, deoxycytidine, and their analog monophosphates by human UMP/CMP kinase is differentially regulated by ATP and magnesium
Phosphorylation of cytidine, deoxycytidine, and their analog monophosphates by human UMP/CMP kinase is differentially regulated by ATP and magnesium
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DOI：
10.1124/mol.104.006098
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发表时间：
2005-03-01
影响因子：
3.6
通讯作者：
Cheng, YC
中科院分区：
文献类型：
--
作者：
Hsu, CH;Liou, JY;Cheng, YC
人UMP/CMP激酶（cytidylate kinase; EC 2.7.4.14）负责CMP、UMP和脱氧胞苷单磷酸（dCMP）的磷酸化，并在嘧啶类似物的激活中起重要作用，其中一些在临床上是有用的抗癌或抗病毒药物。先前使用重组或高度纯化的人UMP/CMP激酶的动力学数据表明，dCMP以及嘧啶类似物单磷酸盐比CMP或UMP对该酶的底物差得多。这意味着必须涉及其他未确定的机制，才能使该酶在细胞内磷酸化dCMP或嘧啶类似物单磷酸成为可能。在这里，我们重新评估了人重组人UMP/CMP激酶磷酸化dCMP和CMP的最佳反应条件（简称dCMPK和CMPK活性）。我们发现ATP和镁是该酶激酶活性的重要调节因子。游离镁增强dCMPK活性，抑制CMPK活性。另一方面，游离ATP或过量的ATP/镁抑制dCMPK反应，但不抑制CMPK反应。ATP或镁对dCMPK和CMPK活性的差异调节也见于其他2'-脱氧嘧啶类似物单磷酸（脱氧尿苷单磷酸，5-氟脱氧尿苷单磷酸，1- β -d -阿拉伯糖单磷酸胞嘧啶和单磷酸吉西他滨）与它们的核糖对应物（UMP和5-氟尿嘧啶单磷酸），以类似的方式。这些数据表明，人UMP/CMP激酶对dCMP和CMP的活性位点不可能相同。此外，酶抑制研究表明，CMP可以以非竞争性方式抑制dCMP的磷酸化，其K-i值远高于其自身的K-m值。因此，我们提出了人类UMP/CMP激酶磷酸化作用的新模型。
Human UMP/CMP kinase (cytidylate kinase; EC 2.7.4.14) is responsible for phosphorylation of CMP, UMP, and deoxycytidine monophosphate (dCMP) and also plays an important role in the activation of pyrimidine analogs, some of which are clinically useful anticancer or antiviral drugs. Previous kinetic data using recombinant or highly purified human UMP/CMP kinase showed that dCMP, as well as pyrimidine analog monophosphates, were much poorer substrates than CMP or UMP for this enzyme. This implies that other unidentified mechanisms must be involved to make phosphorylation of dCMP or pyrimidine analog monophosphates inside cells by this enzyme possible. Here, we reevaluated the optimal reaction conditions for human recombinant human UMP/CMP kinase to phosphorylate dCMP and CMP (referred as dCMPK and CMPK activities). We found that ATP and magnesium were important regulators of the kinase activities of this enzyme. Free magnesium enhanced dCMPK activity but inhibited CMPK activity. Free ATP or excess ATP/magnesium, on the other hand, inhibited dCMPK but not CMPK reactions. The differential regulation of dCMPK versus CMPK activities by ATP or magnesium was also seen in other 2'-deoxypyrimidine analog monophosphates (deoxyuridine monophosphate, 5-fluorodeoxyuridine monophosphate, 1-beta-D-arabinofuranosylcytosine monophosphate, and gemcitabine monophosphate) versus their ribose-counterparts (UMP and 5-fluorouridine monophosphate), in a similar manner. The data suggest that the active sites of human UMP/CMP kinase for dCMP and for CMP cannot be identical. Furthermore, enzyme inhibition studies demonstrated that CMP could inhibit dCMP phosphorylation in a noncompetitive manner, with K-i values much higher than its own K-m values. We thus propose novel models for the phosphorylation action of human UMP/CMP kinase.