对一种含胸苷酸转移酶的核苷四磷酸进行的机制评估。

Mechanistic evaluation of a nucleoside tetraphosphate with a thymidylyltransferase.

作者信息

Forget Stephanie M, Smithen Deborah A, Jee Alison, Jakeman David L

机构信息

Department of Chemistry, Dalhousie University , P.O. Box 15000, Halifax, Canada B3H 4R2.

出版信息

Biochemistry. 2015 Mar 3;54(8):1703-7. doi: 10.1021/bi501438p. Epub 2015 Feb 16.

DOI:10.1021/bi501438p

PMID:25647009

Abstract

Pyrimidine polyphosphates were first detected in cells 5 decades ago; however, their biological significance remains only partially resolved. Such nucleoside polyphosphates are believed to be produced nonspecifically by promiscuous enzymes. Herein, synthetically prepared deoxythymidine 5'-tetraphosphate (p4dT) was evaluated with a thymidylyltransferase, Cps2L. We have identified p4dT as a substrate for Cps2L and evaluated the reaction pathway by analysis of products using high-performance liquid chromatography, liquid chromatography and tandem mass spectrometry, and 31P nuclear magnetic resonance spectroscopy. Product analysis confirmed production of dTDP-Glc and triphosphate (P3) and showed no trace of dTTP-Glc and PPi, which could arise from alternative pathways for the reaction mechanism.

摘要

嘧啶多磷酸盐在50年前首次在细胞中被检测到；然而，它们的生物学意义仍仅得到部分解析。这类核苷多磷酸盐被认为是由混杂酶非特异性产生的。在此，我们用胸苷酰转移酶Cps2L对合成制备的脱氧胸苷5'-四磷酸（p4dT）进行了评估。我们已确定p4dT是Cps2L的一种底物，并通过使用高效液相色谱、液相色谱和串联质谱以及31P核磁共振光谱分析产物来评估反应途径。产物分析证实了dTDP-Glc和三磷酸（P3）的生成，且未检测到可能由反应机制的替代途径产生的dTTP-Glc和焦磷酸（PPi）的痕迹。