Mechanisms on Allosteric Influence on Enzymes Activity

描述（由申请人提供）： 对代谢控制具有根本重要性的调节动机是代谢物对酶活性的变构修饰。这项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。磷酸果糖激酶（PFK）来源于E. coli和B.将嗜热脂肪菌作为模型系统进行研究。这些酶是同源四聚体，每个亚基含有单个活性位点和单个变构位点。尽管这种相对简单的组成，10个独特的成对变构相互作用可以确定。已经产生了这些酶的杂合形式，其分离了单独的变构相互作用，所述变构相互作用共同起作用以产生tetramic酶的调节性质。我们建议调查的变构性能显示的每一个这些杂交，因为在每种情况下的功能相互作用的定义的性质的分子基础。通过建立大肠杆菌的表达菌株，将改进用于产生这些杂交体的程序。不具有PFK基因的螺旋，并通过在同一细胞中共表达两种亲本蛋白来改进体内产生杂交体的程序。提出了一种策略，用于隔离每个2：2的混合，这将允许每个独特的同向性相互作用的隔离和演示如何多个相互作用联合收割机结合在一个二聚体的理论框架内。将通过系统地使用定点诱变来定位在显示单一相互作用的每个杂交体中传递变构影响的推定管道。同时，将用X射线晶体学和时间分辨荧光光谱学研究变构通信的结构基础。后一种技术应该产生关于蛋白质的动态特性的信息，这将补充从晶体学获得的结构信息。最后从两个E.线圈和B。将仔细比较底物和变构配体类似物如何触发它们的变构行为。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特异性的差异有关。

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.