Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, MA 02467, USA.
Biochemistry. 2012 Sep 11;51(36):7128-37. doi: 10.1021/bi300920m. Epub 2012 Aug 24.
Escherichia coli aspartate transcarbamoylase (ATCase) allosterically regulates pyrimidine nucleotide biosynthesis. The enzyme is inhibited by CTP and can be further inhibited by UTP, although UTP alone has little or no influence on activity; however, the mechanism for the synergistic inhibition is still unknown. To determine how UTP is able to synergistically inhibit ATCase in the presence of CTP, we determined a series of X-ray structures of ATCase·nucleotide complexes. Analysis of the X-ray structures revealed that (1) CTP and dCTP bind in a very similar fashion, (2) UTP, in the presence of dCTP or CTP, binds at a site that does not overlap the CTP/dCTP site, and (3) the triphosphates of the two nucleotides are parallel to each other with a metal ion, in this case Mg(2+), coordinated between the β- and γ-phosphates of the two nucleotides. Kinetic experiments showed that the presence of a metal ion such as Mg(2+) is required for synergistic inhibition. Together, these results explain how the binding of UTP can enhance the binding of CTP and why UTP binds more tightly in the presence of CTP. A mechanism for the synergistic inhibition of ATCase is proposed in which the presence of UTP stabilizes the T state even more than CTP alone. These results also call into question many of the past kinetic and binding experiments with ATCase with nucleotides as the presence of metal contamination was not considered important.
大肠杆菌天冬氨酸转氨甲酰酶（ATCase）通过变构调节嘧啶核苷酸的生物合成。该酶被 CTP 抑制，并且可以被 UTP 进一步抑制，尽管 UTP 单独对活性几乎没有影响；然而，协同抑制的机制仍不清楚。为了确定在 CTP 存在的情况下 UTP 如何能够协同抑制 ATCase，我们确定了一系列 ATCase·核苷酸复合物的 X 射线结构。X 射线结构的分析表明：（1）CTP 和 dCTP 以非常相似的方式结合；（2）UTP 在 dCTP 或 CTP 的存在下，结合在不与 CTP/dCTP 位点重叠的位点上；（3）两个核苷酸的三磷酸彼此平行，并且在这种情况下有一个金属离子（例如 Mg(2+)）与两个核苷酸的β-和γ-磷酸配位。动力学实验表明，需要金属离子（例如 Mg(2+)）的存在才能进行协同抑制。这些结果共同解释了为什么 UTP 的结合可以增强 CTP 的结合，以及为什么在 CTP 存在的情况下 UTP 结合得更紧密。提出了一种 ATCase 协同抑制的机制，其中 UTP 的存在比 CTP 单独存在更能稳定 T 态。这些结果还对过去许多涉及 ATCase 和核苷酸的动力学和结合实验提出了质疑，因为当时没有考虑到金属污染的存在非常重要。
