MECHANISM OF ALLOSTERIC INFLUENCE ON ENZYME ACTIVITY

对新陈代谢控制至关重要的一个调控主题是 代谢产物对酶活性的变构修饰。《长河》 本次竞争性续签申请的任期目标仍然是 加深我们对变构配体的作用机理的理解 能够通过结合到一个 从活性部位去除的酶。我们特别感兴趣的是 变构配体通过改变来实现其作用的体系 酶对其底物的亲和力。 将研究三种不同的变构酶：磷酸果糖激酶 (Pfk)来自大肠杆菌，pfk来自嗜热脂肪芽孢杆菌；以及 大肠杆菌的氨基甲酰磷酸合成酶(CPS)。这其中的每一个 酶现在被克隆并在各种大肠杆菌菌株中过度表达，因此 大量的酶可用于生物物理，热力学， 以及动力学研究。这些菌株还提供了产生 定点突变体。通过共同研究这些酶，一个更大的 对变构酶的一般性质的理解应该 比狭隘地专注于特定的目标更加坦率 单一酶所呈现的机械性问题。 四种新的实验方法将被应用于这些研究 酶：定位的频域荧光光谱学 诱变、等温微量热法和高静水压 申请。有了这些技术，热和热的意义 熵对耦合自由能的贡献，它定量地 定义了变构效应的性质和大小，意志 被探索。尤其是为什么有些酶似乎 展示了由熵变化控制的耦合自由能 激活剂和抑制剂，而对于其他酶来说，没有偶联 能量是由变化的热能决定的，将会被解决。它是 假设配体诱导的动力学微扰 酶的结构可能对偶联的熵分量有贡献 自由能。如果是真的，这一假设意味着 变构配体在结构上的作用可能是看不见的 酶-配体络合物的描绘，例如由x射线提供的那些 结晶学。

A regulatory motif of fundamental importance to metabolic control is the allosteric modification of enzymatic activity by metabolites. The long term objective of this competitive renewal application continues to be to increase our understanding of the mechanisms by which allosteric ligands are able to modify enzymatic activity through binding to sites on an enzyme removed from the active site. In particular we are interested in systems in which the allosteric ligands achieve their effects by altering the affinity of the enzyme for its substrate. Three different allosteric enzymes will be studied: phosphofructokinase (PFK) from Escherichia coli, PFK from Bacillus stearothermophilus; and carbamoyl phosphate synthetase (CPS) from Escherichia coli. Each of these enzymes is now cloned and overexpressed in various E. coli strains so that copious quantities of enzyme are available for biophysical, thermodynamic, and kinetic studies. The strains also provide the means for generating site-directed mutants. By studying these enzymes in concert, a greater understanding of general properties exhibited by allosteric enzymes should be forthcoming than would result from a narrow focus on specific mechanistic issues presented by a single enzyme. Four new experimental approaches will be applied to the study of these enzymes: frequency-domain fluorescence spectroscopy, site-directed mutagenesis, isothermal microcalorimetry, and high hydrostatic pressure application. With these techniques the significance of the enthalpy and entropy contributions to the coupling free energy, which quantitatively defines both the nature and the magnitude of the allosteric effect, will be explored. In particular the question of why some enzymes seem to exhibit coupling free energies that are dominated by entropy changes for both activators and inhibitors, whereas for other enzymes coupling free energies are dominated by changes in enthalpy will be addressed. It is hypothesized that ligand-induced perturbations of the dynamics of the enzyme structure may contribute to the entropy component of the coupling free energy. If true, this hypothesis implies that much of what an allosteric ligand does upon binding might be invisible to structural depictions of enzyme-ligand complexes such as those afforded by x-ray crystallography.