MECHANISMS ON ALLOSTERIC INFLUENCES ON ENZYMES ACTIVITY

代谢控制的一个重要调控基序是代谢产物对酶活性的变构修饰。这项研究计划的长期目标是了解变构调节酶活性的分子基础。特别是，我们对变构配体通过改变酶对其底物的亲和力来实现其作用的系统感兴趣。三种不同的变构酶将作为模型系统进行研究：来自大肠杆菌的磷酸果糖激酶(PFK)、来自嗜热脂肪杆菌的PFK和来自大肠杆菌的氨基甲酰磷酸合成酶(CPS)。在具体目标I中，将构建一系列来自大肠杆菌和嗜热脂肪杆菌的PFK突变杂交种，其中3个亚基上的结合位点将被排列在结合位点上的正电荷残基的突变所敲除。剩下的野生型亚基将产生一个高亲和力底物结合位点和一个高亲和力变构结合位点。通过改变突变的残基，4个位点之间的相互作用将被分离并单独研究。特定目的II需要系统地评价变构作用和结合专一性之间的关系。我们将对来自大肠杆菌和嗜热脂肪杆菌的CPS和PFK的底物和变构配体类似物进行评估，以验证我们的假设，即如果建立变构效应的耦合自由能是熵控制的，变构配体可以对结合特异性产生反转效应。在具体目标三中，我们将评估变构配体的平衡效应如何表现在配体结合和解离的动力学中，以便更准确地描绘所涉及的实际微扰。将特别注意底物和变构配体似乎独立结合的情况，但从基本热力学中有证据表明，这两个位置可能是‘默默’耦合的。这些研究应该为变构配体实现其作用的具体分子机制提供重要的见解。这些信息最终将在医学工作中被证明是有用的，例如药物设计，这需要对配体结合的结构后果和低于蛋白质-配体相互作用的特异性的性质有精确的了解。

A regulatory motif of fundamental importance to metabolic control is the allosteric modification of enzymatic activity by metabolites. The long-term goal of this research program is to understand the molecular basis for allosteric regulation of enzyme activity. 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 as model systems: phosphofructokinase (PFK) from E. coli, PFK from B. stearothermophilus; and carbamoyl phosphate synthetase (CPS) from E. coli. In Specific Aim I, a series of mutant hybrids of PFK from both E. coli and B. stearothermophilus will be constructed in which the binding sites on 3 of the subunits will have been knocked out with mutations of the positively charged residues that line the binding sites. The remaining wild-type subunit will create a single high-affinity substrate binding site and a single high affinity allosteric binding site. By varying which residues are mutated, each of the 4 site-site interactions will be isolated and studied individually. Specific Aim II entails a systematic evaluation of the relationship between allosteric action and binding specificity. Substrate and allosteric ligand analogs of CPS and PFK from both E. coli and B. stearothermophilus will be evaluated to test our hypothesis that allosteric ligands can have an inverted effect on binding specificity if the coupling free energy that establishes the allosteric effect is entropy-dominated. In Specific Aim III we will evaluate how the equilibrium effects of allosteric ligands are manifested in kinetics of ligand binding and dissociation in order to paint a more accurate picture of the actually perturbation that is involved. Particular attention will be paid to those instances where the substrate and the allosteric ligand appear to bind independently but where there is evidence from the underlying thermodynamics that the sites may be 'silently' coupled. These investigations should provide significant insight into the specific molecular mechanisms by which allosteric ligands achieve their effects. This information will ultimately prove useful in medical efforts, such as drug design, which require a precis knowledge of the structural consequences of ligand binding and of the properties that underly the specificity of protein-ligand interactions.