Pasti Claudia, Gallois-Montbrun Sarah, Munier-Lehmann Hélène, Veron Michel, Gilles Anne-Marie, Deville-Bonne Dominique
Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 2185, Institut Pasteur, Paris, France.
Eur J Biochem. 2003 Apr;270(8):1784-90. doi: 10.1046/j.1432-1033.2003.03537.x.
UMP-CMP kinase catalyses an important step in the phosphorylation of UTP, CTP and dCTP. It is also involved in the necessary phosphorylation by cellular kinases of nucleoside analogs used in antiviral therapies. The reactivity of human UMP-CMP kinase towards natural substrates and nucleotide analogs was reexamined. The expression of the recombinant enzyme and conditions for stability of the enzyme were improved. Substrate inhibition was observed for UMP and CMP at concentrations higher than 0.2 mm, but not for dCMP. The antiviral analog l-3TCMP was found to be an efficient substrate phosphorylated into l-3TCDP by human UMP-CMP kinase. However, in the reverse reaction, the enzyme did not catalyse the addition of the third phosphate to l-3TCDP, which was rather an inhibitor. By molecular modelling, l-3TCMP was built in the active site of the enzyme from Dictyostelium. Human UMP-CMP kinase has a relaxed enantiospecificity for the nucleoside monophosphate acceptor site, but it is restricted to d-nucleotides at the donor site.
UMP-CMP激酶催化UTP、CTP和dCTP磷酸化过程中的一个重要步骤。它还参与了抗病毒治疗中使用的核苷类似物被细胞激酶进行的必要磷酸化反应。重新研究了人UMP-CMP激酶对天然底物和核苷酸类似物的反应活性。改进了重组酶的表达及酶的稳定性条件。当UMP和CMP浓度高于0.2 mM时观察到底物抑制现象，但dCMP未出现该现象。发现抗病毒类似物l-3TCMP是一种有效的底物，可被人UMP-CMP激酶磷酸化为l-3TCDP。然而，在逆反应中，该酶不催化向l-3TCDP添加第三个磷酸基团，l-3TCDP反而是一种抑制剂。通过分子建模，将l-3TCMP构建在盘基网柄菌酶的活性位点中。人UMP-CMP激酶对核苷单磷酸受体位点具有宽松的对映体特异性，但在供体位点仅限于d-核苷酸。
