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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Substitutions in the aspartate transcarbamoylase domain of hamster CAD disrupt oligomeric structure
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Substitutions in the aspartate transcarbamoylase domain of hamster CAD disrupt oligomeric structure

机译：仓鼠CAD的天冬氨酸转氨甲酰酶结构域的取代破坏寡聚结构。

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摘要

Aspartate transcarbamoylase (ATCase: EC 2.1.3.2) is one of three enzymatic domains of CAD, a protein whose native structure is usually a hexamer of identical subunits. Alanine substitutions for the ATCase residues Asp-90 and Arg-269 were generated in a bicistronic vector that encodes a 6-histidine-tagged hamster CAD. Stably transfected mammalian cells expressing high levels of CAD were easily isolated and CAD purification was simplified over previous procedures. The substitutions reduce the ATCase V_max of the altered CADs by 11-fold and 46-fold, respectively, as well as affect the enzyme's affinity for aspartate. At 25 mM Mg~2+, these substitutions cause the oligomeric CAD to dissociate into mono- mers. Under the same dissociating conditions. incubating the altered CAD with the ATCase substrate carbamoyl phosphate or the bisubstrate analogue N-phosphonacetyl-L-aspartate unexpect- edly leads to the reformation of hexamers. Incubation with the other ATCase substrate. aspartate, has no effect. These results demonstrate that the ATCase domain is central to hexamer for- mation in CAD and suggest that the ATCase reaction mechanism is ordered in the same manner as the Escherichia coli ATCase. Finally, the data indicate that the binding of carbamoyl phosphate induces conformational changes that enhance the interaction of CAD subunits.
机译：天冬氨酸转氨甲酰酶（ATCase：EC 2.1.3.2）是CAD的三个酶结构域之一，CAD是一种天然结构通常为相同亚基的六聚体的蛋白质。在编码6-组氨酸标签的仓鼠CAD的双顺反子载体中产生ATCase残基Asp-90和Arg-269的丙氨酸取代。易于分离表达高水平CAD的稳定转染的哺乳动物细胞，并且与以前的程序相比，简化了CAD纯化。取代使改变的CAD的ATCase V_max分别降低11倍和46倍，并且影响酶对天冬氨酸的亲和力。在25 mM Mg〜2 +时，这些取代导致寡聚CAD解离为单体。在相同的解离条件下。用ATCase底物氨基甲酰基磷酸酯或双底物类似物N-膦酰基乙酰基-L-天门冬氨酸孵育改变的CAD会意外地导致六聚体的重新形成。与其他ATCase底物一起孵育。天冬氨酸，没有作用。这些结果表明，ATCase结构域是CAD中六聚体形成的核心，并表明ATCase反应机理的顺序与大肠杆菌ATCase相同。最后，数据表明氨基甲酰基磷酸酯的结合诱导构象变化，该构象变化增强了CAD亚基的相互作用。

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