Department of Chemistry and Biochemistry, Eastern Washington University, 226 Science Building, Cheney, WA 99004, USA.
Biochimie. 2013 Sep;95(9):1711-21. doi: 10.1016/j.biochi.2013.05.016. Epub 2013 Jun 14.
Inosine triphosphate (ITP) pyrophosphohydrolase, or ITPase, is an intracellular enzyme that is responsible for the hydrolysis of the acidic anhydride bond between the alpha and beta phosphates in ITP, and other noncanonical nucleoside triphosphates, producing the corresponding nucleoside monophosphate and pyrophosphate. This activity protects the cell by preventing noncanonical nucleoside triphosphates from accumulating in (deoxy) nucleoside triphosphate ((d)NTP) pools and/or being integrated into nucleic acids. This enzyme is encoded by the ITPA gene in mammals. It has been reported that Itpa homozygous-null knock-out mice die before weaning and have gross cardiac abnormalities. Additionally, certain variations in the human ITPA gene have been linked to adverse reactions to the immunosuppressive prodrugs azathioprine and 6-mercaptopurine and protection against ribavirin-induced hemolytic anemia. These drugs are bioactivated to form noncanonical nucleoside triphosphates. Human ITPase enzymes engineered to modulate nucleobase specificity may be valuable tools for studying the role of ITPase in heart development and drug metabolism or developing gain-of-function mutants or inhibitory molecules. Based on x-ray crystallography and amino acid sequence data, a panel of putative human ITPase nucleobase specificity mutants has been generated. We targeted eight highly conserved amino acid positions within the ITPase sequence that correspond to amino acids predicted to directly interact with the nucleobase or help organize the nucleobase binding pocket. The ability of the mutants to protect against exogenous and endogenous noncanonical purines was tested with two Escherichia coli complementation assays. Nucleobase specificity of the mutants was investigated with an in vitro biochemical assay using ITP, GTP and ATP as substrates. This methodology allowed us to identify gain-of-function mutants and categorize the eight amino acid positions according to their ability to protect against noncanonical purines as follows: Glu-22, Trp-151 and Arg-178, essential for protection; Phe-149, Asp-152, Lys-172 and Ser-176, intermediate protection; His-177, dispensable for protection against noncanonical purines.
肌苷三磷酸（ITP）焦磷酸水解酶，或 ITPase，是一种细胞内酶，负责水解 ITP 中α和β磷酸之间的酸性酐键，以及其他非规范核苷三磷酸，生成相应的核苷一磷酸和焦磷酸。这种活性通过防止非规范核苷三磷酸在（脱氧）核苷三磷酸（dNTP）池中积累和/或整合到核酸中来保护细胞。这种酶在哺乳动物中由 ITPA 基因编码。据报道，Itpa 纯合敲除的 knockout 小鼠在断奶前死亡，并且心脏有严重的异常。此外，人类 ITPA 基因的某些变异与免疫抑制剂前药巯嘌呤和 6-巯基嘌呤的不良反应以及利巴韦林诱导的溶血性贫血的保护有关。这些药物被生物转化为形成非规范核苷三磷酸。经过工程改造以调节碱基特异性的人类 ITPase 酶可能是研究 ITPase 在心脏发育和药物代谢中的作用或开发功能获得性突变体或抑制性分子的有价值的工具。基于 X 射线晶体学和氨基酸序列数据，已经生成了一组推定的人类 ITPase 碱基特异性突变体。我们针对 ITPase 序列中八个高度保守的氨基酸位置进行了靶向，这些位置对应于预测与碱基直接相互作用或帮助组织碱基结合口袋的氨基酸。通过两种大肠杆菌互补测定来测试突变体对外源和内源性非规范嘌呤的保护能力。通过使用 ITP、GTP 和 ATP 作为底物的体外生化测定来研究突变体的碱基特异性。这种方法使我们能够识别功能获得性突变体，并根据它们对非规范嘌呤的保护能力对八个氨基酸位置进行分类，如下所示：Glu-22、Trp-151 和 Arg-178，是保护所必需的；Phe-149、Asp-152、Lys-172 和 Ser-176，中间保护；His-177，对非规范嘌呤的保护可有可无。
