Structural and Biochemical Studies on the Reaction Mechanism of Uridine-Cytidine Kinase
Structural and Biochemical Studies on the Reaction Mechanism of Uridine-Cytidine Kinase
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DOI：
10.1007/s10930-015-9636-8
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发表时间：
2015-12-01
期刊：
影响因子：
3
通讯作者：
Masui, Ryoji
中科院分区：
文献类型：
--
作者：
Tomoike, Fumiaki;Nakagawa, Noriko;Masui, Ryoji
尿苷-胞苷激酶催化嘧啶核苷尿苷和胞苷的磷酸化，在核苷酸代谢中发挥重要作用。然而，这些反应的详细分子机制仍有待阐明。在这里，我们确定了来自嗜热栖热菌 HB8 的尿苷-胞苷激酶与胞苷和 β,γ-亚甲基腺苷 5'-三磷酸（一种不可水解的 ATP 类似物）的三元复合物的结构。底物结合伴随着大量的结构域移动，使得底物结合裂口闭合。 ATP 类似物的末端磷酸二酯键位于胞苷 5'-羟基内联攻击的理想位置。 Asp40 位于胞苷的 5'-羟基附近。该保守残基突变为 Asn 或 Ala 导致酶活性完全丧失，这与 Asp40 作为激活胞苷 5'-羟基的通用碱基的概念一致。活性的 pH 曲线显示表观 pK (a) 值为 7.4。基于该结构，讨论了催化步骤的可能机制。
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 beta,gamma-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.