Thermal Stability of Enzyme I of PEP:Sugar Phosphotransferase System of E. coli

PEP酶I的热稳定性：大肠杆菌糖磷酸转移酶系统

• 批准号: 6109154
• 负责人: ANN GINSBURG
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6109154
• 关键词: Escherichia coli; enzyme complex; enzyme structure; phosphoenolpyruvate; phosphotransferases; protein folding; protein structure function; thermostability

The widespread bacterial phosphoenolpyruvate (PEP) sugar phosphotransferase system (PTS) is capable of carrying out the coupled translocation and phosphorylation of numerous sugars. The system is composed of two cytoplasmic proteins (enzyme I and HPr) that are used for all sugars and sugar-specific proteins known as enzyme II. We have been investigating structure/function relationships of E. coli enzyme I (MW=63,500) and its N-terminal domain (EIN, MW = 28,346), which is inactive in autophosphorylation but is phosphorylated at His 189 by transfer from phospho-HPr. Previously, we found that phosphorylation of the active-site His 189 destabilizes the amino terminal domain of enzyme I, as shown by a 7 deg C decrease in the Tm of the two-state thermal unfolding transition at pH 7.5. A decrease in the conformational stability of the amino terminal domain by phosphorylation of His 189 promotes phosphotransfer to HPr. In order to investigate the effects of phosphorylation on the stability of enzyme I further, we have substituted Ala and Glu for His 189 to produce H189A and H189E mutants of both EIN and the full length enzyme I. These proteins have been expressed and purified. Differential scanning calorimetry (DSC) and temperature-induced changes in circular dichroism (CD) at 222 nm for wild-type (wt: H189-) and mutant EIN proteins have shown two-state unfolding for all three proteins in 10 mM K-phosphate, pH 7.5 buffer with the following parameters: Tm = 57 deg C ([Delta H] = 140 kcal/mol) for wt EIN and Tm = 50 deg C for phosphorylated wt EIN; Tm = 55 deg C ([Delta H] = 130 kcal/mol) for H189A; and Tm = 53 deg C ([Delta H] = 130 kcal/mol) for H189E. Thus, the mutations at His 189 decreased the thermal stability of EIN and the introduction of a negative charge at the active site of EIN was the most destabilizing. Similar studies will be conducted on the same active-site mutants of intact enzyme I. In addition, isothermal titration calorimetry will be used to measure affinity changes for HPr produced by Ala and Glu substitutions for His at position 189. Ultracentrifugation is being used also to measure hydrodynamic properties of mutant and wild-type proteins.

广泛存在的细菌磷酸烯醇丙酮酸 (PEP)糖磷酸转移酶系统（PTS）能够 进行偶联的易位和磷酸化， 许多糖。该系统由两个细胞质 用于所有糖的蛋白质（酶I和HPr）， 称为酶II的糖特异性蛋白质。我们一直 研究E.大肠杆菌酶I (MW= 63，500）及其N-末端结构域（EIN，MW = 28，346）， 其在自磷酸化中无活性，但在 His 189由磷酸-HPr转移而来。此前，我们发现， 活性位点His 189的磷酸化使氨基酸不稳定， 酶I的末端结构域，如由酶I的末端结构域中的7 ° C降低所示。 在pH 7.5下的两态热去折叠转变的Tm。一 氨基末端构象稳定性降低 结构域的磷酸化His 189促进磷酸转移到 HPr.为了研究磷酸化对细胞凋亡的影响， 酶I的稳定性此外，我们用Ala和Glu取代了 His 189产生EIN和EIN的H189 A和H189 E突变体 全长酶I。这些蛋白质已经表达， 提纯差示扫描量热法（DSC）和 222 nm处圆二色性（CD）的温度诱导变化 野生型（wt：H189-）和突变型EIN蛋白的表达 所有三种蛋白质在10 mM磷酸钾中的双态解折叠， pH 7.5缓冲液，参数如下：Tm = 57 ℃ （[Δ H] = 140 kcal/mol），对于wt EIN，Tm = 50 deg C， 磷酸化wt EIN; Tm = 55 ℃（[Δ H] = 130 kcal/mol） 对于H189 A; Tm = 53 ℃（[Δ H] = 130 kcal/mol）， H189E。因此，His 189处的突变降低了细胞的热稳定性。 EIN的稳定性和在 EIN的活性位点是最不稳定的。类似的研究将 进行相同的活性位点突变体的完整酶I。在 此外，等温滴定量热法将用于测量 由Ala和Glu取代产生的HPr的亲和力变化， 他在189号位置。超离心也被用于 测量突变体和野生型蛋白质的流体动力学性质。