Gilles A M, Cristea I, Palibroda N, Hilden I, Jensen K F, Sarfati R S, Namane A, Ughetto-Monfrin J, Bârzu O
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Anal Biochem. 1995 Dec 10;232(2):197-203. doi: 10.1006/abio.1995.0007.
UTP, labeled with 15N and 13C (at all carbon atoms of the ribose moiety), was obtained enzymatically from [15N]uracil and [13C6]glucose. Eleven enzymes and suitable substrates reconstituted a metabolic pathway in which glucose was first transformed to 5-phosphoribosyl-1-pyrophosphate. The latter compound plus uracil yielded UMP in a second step by the reaction catalyzed by uracil phosphoribosyltransferase. UMP was subsequently phosphorylated to the corresponding di- and triphosphate. ATP, required for five phosphorylation reactions, was regenerated from creatine phosphate, whereas NADP+ necessary for the oxidation of glucose 6-phosphate and 6-phosphogluconate was recycled by glutamate dehydrogenase and excess of ammonia and alpha-oxoglutarate. Despite the number and complexity of the enzymatic steps, the synthesis of [15N, 13C]UTP is straightforward with an overall yield exceeding 60%. This method, extended and diversified to the synthesis of all natural ribonucleotides, is a more economical alternative for obtaining nucleic acids for structural analysis by heteronuclear NMR spectroscopy.
用[¹⁵N]尿嘧啶和[¹³C₆]葡萄糖通过酶促反应得到在核糖部分的所有碳原子上都标记有¹⁵N和¹³C的UTP。11种酶和合适的底物重构了一条代谢途径，其中葡萄糖首先转化为5-磷酸核糖-1-焦磷酸。后一种化合物与尿嘧啶在尿嘧啶磷酸核糖转移酶催化的反应中第二步生成UMP。UMP随后被磷酸化生成相应的二磷酸和三磷酸。五个磷酸化反应所需的ATP由磷酸肌酸再生，而6-磷酸葡萄糖和6-磷酸葡萄糖酸氧化所需的NADP⁺则由谷氨酸脱氢酶以及过量的氨和α-酮戊二酸循环利用。尽管酶促步骤数量众多且复杂，但[¹⁵N,¹³C]UTP的合成很简单，总产率超过60%。这种方法经过扩展和多样化可用于合成所有天然核糖核苷酸，是通过异核NMR光谱获得用于结构分析的核酸的更经济的替代方法。
