Domain Stability in Enzyme I of the E. coli PEP:Sugar Phosphotransferase System

广泛存在的细菌磷酸烯醇丙酮酸（PEP）：糖磷酸转移酶系统（PTS）将许多糖的易位和磷酸化偶联。该系统由两种细胞质蛋白（酶I和HPr）组成，它们用于所有糖以及糖特异性的膜结合成分酶II和III。PEP是组氨酸189上I酶的Mg（II）依赖性自磷酸化的磷酸化供体。利用差示扫描量热法（DSC）和远紫外圆二色法（CD）研究了大肠杆菌PTS酶I （63,562 Da亚基）和酶I克隆氨基末端结构域（EIN; 28,346 Da）在pH 7.5下的热稳定性。先前，我们发现活性位点His 189的磷酸化使酶I的氨基末端结构域不稳定约7 c。His 189的磷酸化降低了氨基末端结构域的构象稳定性，促进了磷向HPr （PTS的下一个蛋白质）的转移。为了进一步研究磷酸化对全长酶I和EIN稳定性的影响，我们通过蛋白质生物工程产生了活性位点突变（H189E，预计具有磷酸化形式的特性，和H189A）。野生型EIN（H189）和突变型EIN蛋白在222 nm处的DSC和温度诱导的椭圆率变化表明，这三种蛋白在10 mM K-磷酸盐（和100 mM KCl）、pH 7.5条件下均呈现两态展开，展开焓为140 (160)kcal/mol，热容变化为2.7 (3.3)kcal/(K mol)。EIN（wt）、EIN（H189A）和EIN（H189E）对应的转变温度（Tm）值分别为57（59）、55（58）和53（56）℃。由于构象熵的差异，在pH 7.5条件下，EIN中去磷-、磷-His189和His189取代的整体构象稳定性顺序为His > Ala > Glu > His- p，因此在EIN的活性位点引入负电荷是最不稳定的，而中盐对磷-EIN和EIN（H189E）的屏蔽作用最大。HPr的结合在每个病例中产生了3度的稳定。完整酶I的N端和c端结构域之间的远程相互作用正在研究中。c端结构域对于酶I的二聚化是必需的，初步的沉降平衡研究表明，单体二聚体的平衡受到His 189的磷酸化或在n端结构域189位置的Glu取代His的影响。DSC结果表明，在没有底物的情况下，N端和c端结构域的热展开和再折叠反应是弱耦合的。有了失活EI(H189A)，我们可以研究底物Mg(II)- pep与c端结构域结合对酶i整体稳定性和二聚化电位的影响。我们发现，在Mg(II)- pep存在下，N-和c端结构域的展开紧密耦合，两个结构域随着Tm的大幅增加而明显稳定。此外，通过Mg(II)-PEP结合，EI（H189A）的单体-二聚体缔合常数提高了4-6个数量级。这表明细胞内Mg(II)-PEP的浓度决定了自磷酸化酶I二聚体的数量。

The widespread bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) couples the translocation and phosphorylation of numerous sugars. The system is composed of two cytoplasmic proteins (enzyme I and HPr) that are used for all sugars as well as sugar specific, membrane-bound components known as enzymes II and III. PEP is the phosphoryl donor in the Mg(II)-dependent autophosphorylation of enzyme I on histidine 189.Thermal stabilities of enzyme I (63,562 Da subunit) of the E. coli PTS and a cloned amino terminal domain of enzyme I (EIN; 28,346 Da) have been investigated by differential scanning calorimetry (DSC) and far UV circular dichroism (CD) at pH 7.5. Previously, we showed that phosphorylation of the active-site His 189 destabilizes the amino terminal domain of enzyme I by ca. 7 C. A decrease in the conformational stability of the amino terminal domain by phosphorylation of His 189 promotes phosphotransfer to HPr (the next protein of the PTS). In order to investigate the effects of phosphorylation on the stability of full-length enzyme I and EIN further, we have produced active-site mutations (H189E, expected to have the properties of phosphorylated forms, and H189A) by protein bioengineering. DSC and temperature-induced changes in ellipticity at 222 nm for wild-type EIN(H189) and mutant EIN proteins show two-state unfolding for all three proteins in 10 mM K-phosphate (and 100 mM KCl), pH 7.5 with an unfolding enthalpy of 140 (160) kcal/mol and heat capacity change of 2.7 (3.3) kcal/(K mol). Corresponding transition temperature (Tm) values are 57 (59), 55 (58), and 53 (56) C for EIN(wt), EIN(H189A), and EIN(H189E), respectively. The order of overall conformational stability of dephospho- and phospho-His189 and His189 substitutions in EIN at pH 7.5 is His > Ala > Glu > His-P due to differences in conformational entropy, Thus, the introduction of a negative charge at the active site of EIN is the most destabilizing, and neutral salt has the greatest shielding effect on phospho-EIN and EIN(H189E). The binding of HPr produced a 3 degree stabilization in each case. Long-range interactions between the N- and C-terminal domains of intact enzyme I are being investigated. The C-terminal domain is necessary for dimerization of enzyme I and preliminary sedimentation equilibrium studies suggest that the monomer-dimer equilibrium is affected by phosphorylation of His 189 or by substitution of Glu for His at position 189 in the N-terminal domain. DSC results indicate that thermal unfolding and refolding reactions of the N- and C-terminal domains in the absence of substrates are weakly coupled energetically. Having the inactive EI(H189A) allows us to study the effects of binding the substrate Mg(II)-PEP to the C-terminal domain on the overall stability and dimerization potential of enzyme I. We are finding that in the presence of Mg(II)-PEP, the unfolding of N- and C-terminal domains is tightly coupled and both domains are markedly stabilized with large Tm increases. Moreover, the monomer-dimer association constant of EI(H189A) is increased 4-6 orders of magnitude by Mg(II)-PEP binding. This suggests that intracellular concentrations of Mg(II)-PEP determine the amount of enzyme I dimer for autophosphorylation.