MECHANISMS ON ALLOSTERIC INFLUENCES ON ENZYMES ACTIVITY

变构对酶活性影响的机制

• 批准号: 6385525
• 负责人: GREGORY Duncan REINHART
• 金额: $ 30.12万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-08-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385525
• 关键词: 6 phosphofructokinase; Bacillus stearothermophilus; Escherichia coli; allosteric site; bacterial proteins; carbamoylphosphate synthase; chemical binding; chemical kinetics; conformation; enzyme activity; enzyme structure; gene mutation; thermodynamics

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.

对代谢控制具有根本重要性的调节基序是代谢物对酶活性的变构修饰。 该研究计划的长期目标是了解酶活性变构调节的分子基础。 特别是，我们感兴趣的系统中，变构配体实现其影响，通过改变其底物的酶的亲和力。 本论文将研究三种不同的变构酶作为模型系统：来自大肠杆菌的磷酸果糖激酶（PFK）; coli、来自B的PFK。嗜热脂肪菌（stearothermophilus）;杆菌 在Specific Aim I中，我们从E. coli和B.将构建嗜热脂肪菌，其中3个亚基上的结合位点将已用排列在结合位点上的带正电荷残基的突变敲除。 剩余的野生型亚基将产生单个高亲和力底物结合位点和单个高亲和力变构结合位点。 通过改变突变的残基，将单独分离和研究4个位点间相互作用中的每一个。 特异性目标II需要系统评价变构作用与结合特异性之间的关系。 CPS和PFK的底物和变构配体类似物来自E. coli和B.将评估嗜热脂肪酸菌以检验我们的假设，即如果建立变构效应的偶联自由能是熵主导的，则变构配体可以对结合特异性具有反向效应。 在《特定目标III》中，我们将评估变构配体的平衡效应如何在配体结合和解离动力学中表现出来，以便更准确地描绘所涉及的实际扰动。 将特别注意那些情况下，基板和变构配体似乎独立绑定，但有证据表明，从底层的热力学的网站可能是“沉默”耦合。 这些调查应提供显着的洞察特定的分子机制，变构配体实现其效果。 这些信息最终将被证明是有用的医疗工作，如药物设计，这需要一个精确的知识的配体结合的结构后果和蛋白质-配体相互作用的特异性的基础上的属性。