Mechanisms on Allosteric Influence on Enzymes Activity

描述(由申请人提供)： 对代谢控制至关重要的一个调节动机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。以大肠杆菌和嗜热脂肪芽孢杆菌的磷酸果糖激酶(PFK)为模型系统进行研究。这些酶是同源异构体，每个亚基包含一个活性部位和一个变构部位。尽管组成相对简单，但可以确定10个独特的成对变构相互作用。这些酶的混合形式已经被生产出来，它们分离出单独的变构相互作用，这些相互作用共同作用来产生四元酶的调节特性。由于每种情况下功能相互作用的定义性质，我们建议研究每一种杂化所表现出的变构性质的分子基础。用于生产这些杂交物的程序的改进将通过创造一个既不含pfk基因又不含pfk基因的大肠杆菌表达菌株来开发，并通过在同一细胞中共表达两种亲本蛋白来改进体内生产杂交物的程序。提出了一种隔离每个2：2杂交体的策略，该策略将允许隔离每个独特的各向同性相互作用，并演示多个相互作用如何在二聚体理论框架内结合。在每个表现单一相互作用的杂交种中，变构影响传递的假定管道将通过系统地使用定点突变来定位。同时，还将用X射线结晶学和时间分辨荧光光谱研究变构通讯的结构基础。后一种技术应该产生有关蛋白质的动态性质的信息，这将补充从结晶学获得的结构信息。最后，关于底物和变构配体类似物如何触发它们的变构行为，我们将仔细比较E.coil和B.stearthermophilus的PFK。这些酶在其变构性质的表观热力学基础上不同，这种差异可能与结合专一性的差异有关。

DESCRIPTION (provided by applicant): A regulatory motive 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. Phosphofructokinase(PFK) from E. coli and B. stearothermophilus will be investigated as model systems. These enzymes are homotetramers containing a single active site and a single allosteric site per subunit. Despite this relatively simple composition, 10 unique pair-wise allosteric interactions can be identified. Hybrid forms of these enzymes have been produced that isolate individual allosteric interactions that collectively act to produce the regulatory properties of the tetramic enzyme. We propose to investigate the molecular basis for the allosteric properties displayed by each of these hybrids because of the defined nature of the functional interaction in each case. Improvements in the procedures used to produce these hybrids will be developed by creating an expression strain of E. coil that does not have either gene for PFK and by refining a procedure for producing hybrids in vivo by co-expressing both parent proteins in the same cell. A strategy is proposed for isolating each 2:2 hybrid that will allow for the isolation of each unique homotropic interaction and for the demonstration of how multiple interactions combine within a dimeric theoretical framework. The putative conduit by which allosteric influence is transmitted in each hybrid displaying a single interaction will be located through the systematic use of site-directed mutagenesis. Concurrently the structural basis for the allosteric communication will be studied with X-ray crystallography and time-resolved fluorescence spectroscopy. The latter technique should produce information regarding the dynamic properties of the protein that will complement the structural information obtained from crystallography. Finally PFK from both E. coil and B. stearothermophilus will be carefully compared with respect to how substrate and allosteric ligand analogs trigger their allosteric behavior. These enzymes differ in the apparent thermodynamic basis for their allosteric properties, a difference that might be related to differences in binding specificity.