MECHANISM OF ALLOSTERIC INFLUENCE ON ENZYME ACTIVITY

对代谢控制至关重要的调节基序是 代谢物对酶活性的变构修饰。长 这一竞争性续约申请的长期目标仍然是 增加我们对变构配体 能够通过结合到 酶从活性位点被移除。我们特别感兴趣的是 系统，其中变构配体通过改变 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)来自大肠杆菌的PFK，来自嗜热脂肪芽孢杆菌的PFK;和 来自大肠杆菌的氨甲酰磷酸合成酶（CPS）。 这一切成功都 酶的克隆和过表达在各种E.大肠杆菌菌株， 大量的酶可用于生物物理，热力学， 和动力学研究。这些菌株还提供了产生 定点突变体通过协同研究这些酶， 对变构酶一般性质的理解应 而不是狭隘地关注具体问题， 由单一酶引起的机械问题。 四种新的实验方法将被应用于这些研究 酶：频域荧光光谱，定点 诱变、等温微量热法和高静水压 应用程序.通过这些技术， 熵对耦合自由能的贡献， 定义了变构效应的性质和大小，将 被探索。特别是为什么有些酶似乎 表现出耦合自由能，由熵变主导， 激活剂和抑制剂，而对于其他酶偶联自由 能量主要由焓的变化决定。是 假设，配体诱导的扰动的动力学的 酶的结构可能有助于耦合的熵分量 自由能源如果是真的，这一假设意味着， 变构配体在结合时可能对结构不可见 酶-配体复合物的分离，例如通过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.