Studies on the Mechanism and Control of Enzyme Action

9985565腺苷琥珀酸合成酶是一种普遍存在的酶，它参与两条重要的代谢途径，即嘌呤核苷酸循环和腺嘌呤核苷酸的从头合成。前者维持活跃肌肉组织中的ATP水平，而后者为RNA和DNA生物合成提供必要的前体。即使在缺乏新的嘌呤生物合成途径的生物体中，腺苷琥珀酸合成酶仍然在腺核苷酸的挽救机制中发挥着核心作用。本研究旨在详细了解来自大肠杆菌和小鼠的合成酶的结构与功能的关系。主要关注的是腺苷琥珀酸合成酶的单体-二聚体亚基平衡及其对酶活性的影响。合成酶的底物之一IMP是否诱导从不活跃的单体到活跃的合成酶二聚体的转变？如果是这样的话，这种现象是所有合成酶的普遍调节机制吗？此外，严格的效应物影响大肠杆菌酶活性的机制尚不清楚。这种严格的效应是大肠杆菌对饥饿等应激条件的反应，导致鸟嘌呤核苷酸水平升高，这可能会抑制腺苷琥珀酸合成酶和许多其他酶。腺苷琥珀酸合成酶的化学作用是否增强了某些严格效应器的抑制作用？严格效应器是否通过单一机制抑制合成酶？合成酶催化的反应对必需金属阳离子是二级反应，但在晶体结构中只有一个与GDP相关的镁离子存在。推测第二个镁离子与L-天冬氨酸结合。将设计实验来测试L-天冬氨酸的α-羧基是否是第二个镁离子结合所必需的识别元件。合成酶催化其整个反应为两个部分反应的序列。实验将确定6-磷酰亚胺的合成(第一反应)或腺苷琥珀酸酯的合成(第二反应)是否为限速反应。这项拟议的研究将采用多种物理生物化学技术，包括X射线结晶学、31P核磁共振、分析超速离心法和动力学。这些研究将使用重组大肠杆菌和/或小鼠合成酶，在大肠杆菌中表达并从大肠杆菌中分离出来。为了测试几种结构功能假说，我们将构建大肠杆菌和小鼠合成酶的特定突变体。

9985565FrommAdenylosuccinate synthetase, a ubiquitous enzyme, participates in two important metabolic pathways, the purine nucleotide cycle and the de novo biosynthesis of adenine nucleotides. The former maintains ATP levels in active muscle tissue, whereas the latter provides essential precursors for RNA and DNA biosynthesis. Even in organisms that lack a pathway for de novo purine biosynthesis, adenylosuccinate synthetase still plays a central role in salvage mechanisms for adenine nucleotides. This research seeks a detailed understanding of the structure-function relationships of the synthetases from Escherichia coli and from mouse. Of central interest is the monomer-dimer subunit equilibrium of adenylosuccinate synthetases and its effect on enzyme activity. Does IMP, one of the substrates of the synthetase, induce a transition from inactive monomers to active synthetase dimers, and if so, is this phenomenon a universal mechanism of regulation for all synthetases? In addition, the mechanism by which stringent effectors influence activity of the E. coli enzyme is unsettled. The stringent effect is a response by E. coli to conditions of stress, such as starvation, resulting in elevated levels of guanine nucleotides, which putatively inhibit adenylosuccinate synthetase and many other enzymes. Is inhibition by some stringent effectors enhanced by the chemical action of adenylosuccinate synthetase, and do stringent effectors inhibit the synthetase by a single mechanism? The synthetase-catalyzed reaction is second order with respect to the essential metal cation, yet in crystal structures only one Mg2+ is present, associated with GDP. The second Mg2+ binds putatively to L-aspartate. Experiments will be designed to test whether the a-carboxyl group of L-aspartate is an essential recognition element for the binding of the second Mg2+. The synthetase catalyzes its overall reaction as a sequence of two partial reactions. Experiments will determine whether the synthesis of 6-phosphoryl-IMP (first reaction) or the synthesis of adenylosuccinate (second reaction) is rate limiting. The proposed research will employ a variety of techniques in physical biochemistry, including X-ray crystallography, 31P NMR, analytical ultracentrifugation, and kinetics. The studies will employ recombinant E. coli and/or mouse synthetases, expressed in and isolated from E. coli. Specific mutants of the E. coli and mouse synthetases will be constructed in order to test several structure-function hypotheses.