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

广泛存在的细菌磷酸烯醇式丙酮酸(PEP)：糖磷酸转移酶系统(PTS)偶联了许多糖的转位和磷酸化。该系统由两个细胞质蛋白(酶I和HPR)以及糖特异的膜结合组分组成，称为酶II和酶III。PEP是依赖镁(II)的酶I对组氨酸189的自磷酸化反应的磷酸供体。用差示扫描量热法(DSC)和远紫外圆二色谱(CD)研究了大肠杆菌PTS的酶I(63,562 Da亚基)和酶I的氨基末端结构域(ein；28,346 Da)在pH 7.5时的热稳定性。此前，我们发现活性部位His 189的磷酸化使酶I的氨基末端结构域不稳定约7C。通过磷酸化His 189降低氨基末端结构域的构象稳定性，促进磷酸转移到HPR(PTS的下一种蛋白)。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，预期具有磷酸化形式的性质，以及H189A)。DSC和温度诱导的野生型eIN(H189)和突变型eIN蛋白在222 nm处的椭圆度变化显示，这三种蛋白在10 mM磷酸二氢钾(和100 mM KCl)、pH 7.5、展开热容为2.7(3.3)kcal/(Kmo1)、pH 7.5的条件下均呈两态展开。对于EIN(Wt)、EIn(H189A)和Ein(H189E)，相应的转变温度(Tm)值分别为57(59)、55(58)和53(56)℃。在pH 7.5时，由于构象熵的差异，EIN中脱磷和磷酸His189和His189取代的总体构象稳定性顺序为His&gt；Ala&Gt；Glu&Gt；His-P，因此，在EIN的活性中心引入负电荷是最不稳定的，中性盐对磷酸-Ein和Ein(H189E)的屏蔽作用最大。HPR的结合在每一种情况下都产生了3度的稳定性。完整的酶I的N-末端和C-末端结构域之间的长距离相互作用正在研究中。C-末端结构域是酶I二聚化所必需的，初步的沉降平衡研究表明，单体-二聚体平衡受到His 189的磷酸化或N-末端结构域189位的Glu取代His的影响。DSC结果表明，在没有底物的情况下，N-末端和C-末端结构域的热去折叠和复性反应是弱耦合的。有了不活跃的EI(H189A)，我们可以研究底物MG(II)-PEP与C-末端结构域结合对酶I的整体稳定性和二聚化潜力的影响。我们发现，在Mg(II)-PEP的存在下，N-末端和C-末端结构域的展开紧密耦合，两个结构域都明显稳定，Tm增加很大。此外，Mg(II)-PEP结合使EI(H189A)的单体-二聚体缔合常数增加了4-6个数量级。这表明，细胞内镁(II)-PEP的浓度决定了用于自动磷酸化的酶I二聚体的量。

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.