Hsu Chih-Hung, Liou Jieh-Yuan, Dutschman Ginger E, Cheng Yung-Chi
Department of Pharmacology, Yale University School of Medicine, 333 Cedar St., SHM B226, New Haven, CT 06520, USA.
Mol Pharmacol. 2005 Mar;67(3):806-14. doi: 10.1124/mol.104.006098. Epub 2004 Nov 18.
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 Ki values much higher than its own Km values. We thus propose novel models for the phosphorylation action of human UMP/CMP kinase.
人尿苷一磷酸/胞苷一磷酸激酶（胞苷酸激酶；EC 2.7.4.14）负责胞苷一磷酸（CMP）、尿苷一磷酸（UMP）和脱氧胞苷一磷酸（dCMP）的磷酸化，并且在嘧啶类似物的活化过程中也发挥着重要作用，其中一些嘧啶类似物是临床上有用的抗癌或抗病毒药物。先前使用重组或高度纯化的人尿苷一磷酸/胞苷一磷酸激酶的动力学数据表明，dCMP以及嘧啶类似物单磷酸盐作为该酶的底物，比CMP或UMP差得多。这意味着必然涉及其他未明确的机制，以使该酶能够在细胞内对dCMP或嘧啶类似物单磷酸盐进行磷酸化。在此，我们重新评估了重组人尿苷一磷酸/胞苷一磷酸激酶磷酸化dCMP和CMP（分别称为dCMPK和CMPK活性）的最佳反应条件。我们发现ATP和镁是该酶激酶活性的重要调节剂。游离镁增强dCMPK活性，但抑制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活性的差异调节在其他2'-脱氧嘧啶类似物单磷酸盐（脱氧尿苷一磷酸、5-氟脱氧尿苷一磷酸、1-β-D-阿拉伯呋喃糖基胞嘧啶一磷酸和吉西他滨一磷酸）与其核糖对应物（UMP和5-氟尿苷一磷酸）中也以类似方式出现。数据表明，人尿苷一磷酸/胞苷一磷酸激酶针对dCMP和CMP的活性位点不可能相同。此外，酶抑制研究表明，CMP可以非竞争性方式抑制dCMP磷酸化，其抑制常数（Ki）值远高于其自身的米氏常数（Km）值。因此，我们提出了人尿苷一磷酸/胞苷一磷酸激酶磷酸化作用的新模型。
