Department of Chemistry, Boston College , Merkert Chemistry Center, 2609 Beacon Street, Chestnut Hill, MA 02467 U.S.A.
Biochemistry. 2013 Nov 12;52(45):8036-47. doi: 10.1021/bi401205n. Epub 2013 Oct 31.
For nearly 60 years, the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and five X-ray structures determined in the absence and presence of a Mg(2+) concentration within the physiological range. In the presence of 2 mM divalent cations (Mg(2+), Ca(2+), Zn(2+)), CTP does not significantly inhibit the enzyme, while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor of ATCase in vivo is the combination of CTP, UTP, and a divalent cation, and the actual allosteric activator is a divalent cation with ATP or ATP and GTP. The structural data reveals that two NTPs can bind to each allosteric site with a divalent cation acting as a bridge between the triphosphates. Thus, the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal that the catalytic chains undergo essentially no alternations; however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal region of the regulatory chains exists in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed.
近 60 年来，大肠杆菌天冬氨酸转氨甲酰酶（ATCase）的 ATP 激活和 CTP 抑制一直是变构调节的教科书范例。我们呈现了动力学数据和五个在生理范围内缺乏和存在镁（Mg）2+浓度的 X 射线结构。在存在 2mM 二价阳离子（Mg2+、Ca2+、Zn2+）的情况下，CTP 不会显著抑制酶，而 ATP 的变构激活增强。数据表明，ATCase 体内的实际变构抑制剂是 CTP、UTP 和二价阳离子的组合，而实际的变构激活剂是带有 ATP 或 ATP 和 GTP 的二价阳离子。结构数据显示，两个 NTP 可以与每个变构位点结合，二价阳离子在三磷酸之间起桥接作用。因此，ATCase 的调节比以前认为的要复杂得多，这使得许多以前的研究受到质疑。X 射线结构表明，催化链基本上没有变化；然而，调节链的几个区域发生了显著的结构变化。最显著的是，调节链的 N 端区域在酶的变构激活和抑制形式中存在不同的构象。在这里，提出了一个新的变构调节模型。
