The Biology Of Cyclic Nucleotides In E Coli

对大肠杆菌糖运输系统--磷酸烯醇式丙酮酸：糖磷酸转移酶系统(PTS)的蛋白质组分的结构和调控的研究仍在继续。PTS的第一组分(酶I，EI)被活性部位组氨酸上的磷酸烯醇式丙酮酸磷酸化，该磷酸化基团可以转移到第二组分的活性部位(HPR)。EI包含两个域。分离得到的重组螺旋结构域可与HPR结合。用分离的重组螺旋结构域和缺失该结构域的构建体进行的体外重组实验表明，PEP可以恢复自磷酸化。在氨基末端和羧基末端结构域的重组过程中，也恢复了磷酸转移活性。利用生物物理方法对EI活性部位的突变进行了研究，以确定构象稳定性。构象稳定性大小顺序为：His&gt；Ala&gt；Glu&gt；His-PO3。HPR与活性位点突变体的结合不受温度的影响，亲和常数基本相同。一种能够接受P-HPR的磷酰基的PTS蛋白被命名为IIAglc。该蛋白还在调节大肠杆菌中其他糖运输系统的活性方面发挥作用。通过使用先前描述的直接结合实验，鉴定了IIAglc表面与乳糖渗透酶结合的区域。磺基琥珀酰亚胺乙酸酯处理IIAglc使赖氨酸残基乙酰化，降低了与乳糖渗透酶的相互作用程度。为了定位参与调控相互作用的IIAglc上的赖氨酸残基，对选定的赖氨酸残基进行了突变。将九种不同的赖氨酸转化为谷氨酸，得到的蛋白质仍然能够从HPR中接受磷酰化。除Lys69外，所有修饰蛋白均能与乳糖通透酶结合。提出了包括Lys69在内的IIAglc与乳糖渗透酶相互作用的表面区模型。HPR是一种多功能蛋白，作为PTS中的磷酸化载体，它可以与EI和IIAglc相互作用。此外，它还是糖原磷酸化酶的变构调节剂。由于与多种蛋白质相互作用的HPR表面的性质以前是未知的，所以这些相互作用是通过核磁共振进行研究的。测定了15N均一标记的HPR的主链和侧链酰胺1H和15N核在存在和不存在天然丰富的糖原磷酸化酶、IIAglc或EI的氨基末端结构域时的化学位移变化。将这些化学位移扰动映射到HPR的三维结构上，可以识别HPR上与这些蛋白质相互作用的结合表面。映射的界面非常相似，表明HPR在与其合作伙伴结合时使用了相似的表面。

Structural and regulatory studies on protein components of the Escherichia coli (E. coli) sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS) continued. The first component of the PTS (enzyme I, EI) is phosphorylated by phosphoenolpyruvate on an active site histidine and that phosphoryl group can be transferred to the active site of the second component (HPr). EI contains two domains. The isolated recombinant helical domain was shown to bind to HPr. In vitro reconstitution experiments with isolated recombinant helical domain and a construct deficient in that domain showed that it was possible to recover autophosphorylation by PEP. Phosphotransfer activity was also recovered in reconstitution of the aminoterminal and carboxyterminal domains. Mutations at the active site of EI were engineered and studied by biophysical methods for determination of conformational stability. The order of conformational stability is His>Ala>Glu>His-PO3. The binding of HPr to active-site mutants is temperature-independent with about the same affinity constant. One of the PTS proteins that can accept a phosphoryl group from P-HPr is named IIAglc. This protein also plays a role in the regulation of activity of other sugar transport systems in E. coli. By using a previously described direct binding assay, a region on the surface of IIAglc that interfaces with lactose permease was characterized. Actetylation of lysine residues by sulfosuccinimidyl acetate treatment of IIAglc reduced the degree of interaction with lactose permease. To localize the lysine residues on IIAglc that are involved in the regulatory interaction, selected lysine residues were mutagenized. Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr. Except for Lys69, all the modified proteins were able to bind to lactose permease. A model for the region of the surface of IIAglc, including Lys69, that interacts with lactose permease was proposed. HPr is a multifunctional protein; as a phosphocarrier in the PTS, it interacts with both EI and IIAglc. In addition it is an allosteric regulator of glycogen phosphorylase. Because the nature of the surface of HPr that interacts with this multiplicity of proteins was previously undefined, those interactions were investigated by NMR. The chemical shift changes of the backbone and side chain amide 1H and 15N nuclei of uniformly 15N-labeled HPr in the presence and absence of natural abundance glycogen phosphorylase, IIAglc or the aminoterminal domain of EI were determined. Mapping those chemical shift perturbations on the three-dimensional structure of HPr permitted the identification of the binding surface on HPr for interaction with those proteins. The mapped interfaces are remarkably similar, indicating that HPr employs a similar surface in binding to its partners.