Uridine–cytidine kinase catalyzes phosphorylation of the pyrimidine nucleosides uridine and cytidine and plays an important role in nucleotide metabolism. However, the detailed molecular mechanism of these reactions remains to be elucidated. Here, we determined the structure of the ternary complex of Uridine–cytidine kinase from Thermus thermophilus HB8 with both cytidine and β,γ-methyleneadenosine 5′-triphosphate, a non-hydrolysable ATP analogue. Substrate binding is accompanied by substantial domain movement that allows the substrate-binding cleft to close. The terminal phosphodiester bond of the ATP analogue is in an ideal location for an inline attack of the 5′-hydroxyl group of cytidine. Asp40 is located near the 5′-hydroxyl group of cytidine. Mutation of this conserved residue to Asn or Ala resulted in a complete loss of enzyme activity, which is consistent with the notion that Asp40 acts as a general base that activates the 5′-hydroxyl group of cytidine. The pH profile of the activity showed an apparent pK _a value of 7.4. Based on this structure, a likely mechanism of the catalytic step is discussed.

中文翻译：

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尿苷-胞苷激酶反应机理的结构和生化研究。

尿苷-胞苷激酶催化嘧啶核苷尿苷和胞苷的磷酸化，并在核苷酸代谢中起重要作用。但是，这些反应的详细分子机理仍有待阐明。在这里，我们确定了嗜热栖热菌中尿苷-胞苷激酶三元复合物的结构HB8具有胞苷和β，γ-亚甲基腺苷5'-三磷酸酯（一种不可水解的ATP类似物）。底物结合伴随有实质性的域移动，其允许底物结合裂隙闭合。ATP类似物的末端磷酸二酯键处于胞嘧啶胞苷5'-羟基在线攻击的理想位置。Asp40位于胞苷的5'-羟基附近。该保守残基突变为Asn或Ala导致酶活性的完全丧失，这与Asp40充当激活胞苷的5'-羟基的一般碱基的观点相一致。活性的pH曲线显示出明显的p ķ _一个的7.4值。基于这种结构，讨论了催化步骤的可能机理。