PDF(Pubmed)
Abstract：
Recent clinical data have identified infant patients with lethal ITPA deficiencies. ITPA is known to modulate ITP concentrations in cells and has a critical function in neural development which is not understood. Polymorphism of the ITPA gene affects outcomes for both ribavirin and thiopurine based therapies and nearly one third of the human population is thought to harbor ITPA polymorphism. In a previous site-directed mutagenesis alanine screen of the ITPA substrate selectivity pocket, we identified the ITPA mutant, E22A, as a gain-of function mutant with enhanced ITP hydrolysis activity. Here we report a rational enzyme engineering experiment to investigate the biochemical properties of position 22 ITPA mutants and find that the E22D ITPA has two- and four-fold improved substrate selectivity for ITP over the canonical purine triphosphates ATP and GTP, respectively, while maintaining biological activity. The novel E22D ITPA should be considered as a platform for further development of ITPA therapies.
摘要：
最近的临床数据已经确定了具有致死性ITPA缺陷的婴儿患者。已知ITPA调节细胞中的ITP浓度,并且在神经发育中具有关键功能,这一点尚不清楚。ITPA基因的多态性影响基于利巴韦林和硫嘌呤的疗法的结果,并且近三分之一的人群被认为具有ITPA多态性。在先前的ITPA底物选择性口袋的定点诱变丙氨酸筛选中，我们确定了ITPA突变体,E22A,作为具有增强的ITP水解活性的功能获得突变体。在这里，我们报告了一个合理的酶工程实验，以研究第22位ITPA突变体的生化特性，并发现E22DITPA对ITP的底物选择性比经典嘌呤三磷酸ATP和GTP提高了两倍和四倍，分别,同时保持生物活性。新型E22DITPA应被视为进一步开发ITPA疗法的平台。
