TIME EVOLUTION OF THE ALLOSTERIC TRANSITION OF ASPARTATE TRANSCARBAMOYLASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 来自大肠杆菌的天冬氨酸转氨甲酰基酶以两种构象状态存在，即低活性、低亲和力的T状态和高活性的高亲和力R状态。该酶不仅催化了嘧啶生物合成途径中的第一反应，而且参与了整个代谢途径的速率控制。控制被认为是通过改变T和R形式的比例来实现的。酶的T和R状态在功能和结构上都是不同的。在酶从T状态转换到R状态的过程中，酶经历了大约11°的伸长，并伴随着亚基的同时旋转，这一点可以很容易地用SAXS检测到。我们之前已经提出了一种从T态到R态的协同变构转换机制。我们已经能够使用时间分辨SAXS在SSRL上进行一组实验，以直接跟踪从T态到R态的结构转变的时间进程。这些初步数据表明，在转变过程中形成了一种结构中间体。这一建议是为了在SSRL上增加束流时间，以继续研究酶的变构机制以及各向异性效应如何影响结构从T态到R态的转变。这将是变构酶第一次通过SAXS实时跟踪变构结构变化的时间演化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aspartate transcarbamoylase from E. coli exits in two conformational states, a low-activity low-affinity T state and a high-activity high-affinity R state. The enzyme not only catalyzes the first reaction in the pyrimidine biosynthesis pathway, but is also involved in the control of the rate of this entire metabolic pathway. Control is thought to be achieved by altering the ratio of the T and R forms. The T and R states of the enzyme are both functionally and structurally distinct. During the conversion of the enzyme from the T to the R state, the enzyme undergoes an elongation of appoximately 11 ¿, along with simultaneous rotations of subunits, which can easily be detected by SAXS. We have previously proposed a mechanism for a concerted allosteric transition from the T to the R states. We have been able to perform one set of experiments at SSRL using time-resolved SAXS to directly follow the time course of the structural transition from the T to the R state. These preliminary data suggest that a structural intermediate is formed during the transition. This proposal is for additional beam time at SSRL to continue the investigation into the allosteric mechanism of the enzyme and how the heterotropic effects influence the structural transition from the T to the R state. This will be the first time for an allosteric enzyme that the time evolution of the allosteric structural change will be followed in real time by SAXS.