Interdomain interaction and substrate coupling effects on dimerization and conformational stability of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system
Interdomain interaction and substrate coupling effects on dimerization and conformational stability of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system
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DOI：
10.1021/bi011801x
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发表时间：
2002-01-22
期刊：
影响因子：
2.9
通讯作者：
Ginsburg, A
中科院分区：
文献类型：
--
作者：
Dimitrova, MN;Szczepanowski, RH;Ginsburg, A
细菌PEP：糖磷酸转移酶系统将特定糖的磷酸化和跨膜易位偶联。该途径中的第一种蛋白质酶I（EI）的活性受单体-二聚体平衡调节，其中PEP导致的Mg 2+依赖性自磷酸化需要二聚体。通过在pH 7.5、4和20 ℃下的沉降平衡，在各种条件下测定了去磷酸化和磷酸化EI以及失活突变体EI（H189 E）和EI（H189 A）（其中Glu或Ala取代了活性位点His 189）的二聚常数。同时，这些形式的El的热展开已经通过差示扫描量热法和通过内在的Dahanyl残基荧光的变化来监测。在20 ℃下，与去磷酸化EI和EI（H189 A）相比，磷酸化EI和EI（H189 E）的二聚化常数增加了10倍[类似于2 × 10（6）（M单体）-（1）]。如EI（H189 A）所示，1 mM PEP和2 mM MgCl 2强烈促进二聚化[4或20 ℃时K-A'大于或等于10（8）M-1]，EI（H189 A）不能进行自磷酸化。1 mM PEP和2 mM Mg 2+还一起显著稳定和偶联EI（HI 89 A）的C-和N-末端结构域的解折叠，将C-末端结构域解折叠的转变温度（Tm）增加约18 ℃，将N-末端结构域解折叠的转变温度（Tm）增加约9 ℃，使Tmax等于63 ℃，在45 ℃下给出与3 μ MPEP一致的K-D ′值。单独的PEP也促进EI（H189 A）的二聚化，但对于C-末端结构域解折叠，仅增加T-m，接近5 ℃，而不影响N-末端结构域解折叠，在45 ℃下，在不存在Mg 2+的情况下，对于PEP解离，给出K-D'的估计值为0.2mM。相比之下，磷酸-EI在20 ℃下的二聚常数在不存在和存在5 mM PEP和2 MM MgCl 2的情况下是相同的。因此，与非活性EI（H189 A）的研究从磷酸化的底物结合效应的分离表明，PEP和Mg 2+的细胞内浓度是构象稳定性和二聚化的脱磷酸EI的重要决定因素。
The bacterial PEP:sugar phosphotransferase system couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer-dimer equilibrium where a Mg2+-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI and inactive mutants EI(H189E) and EI(H189A) (in which Glu or Ala is substituted for the active site His189) have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5 and 4 and 20 degreesC. Concurrently, thermal unfolding of these forms of El has been monitored by differential scanning calorimetry and by changes in the intrinsic tryptophanyl residue fluorescence. Phosphorylated EI and EI(H189E) have 10-fold increased dimerization constants [similar to2 x 10(6) (M monomer)-(1)] compared to those of dephospho-EI and EI(H189A) at 20 degreesC. Dimerization is strongly promoted by 1 mM PEP with 2 mM MgCl2 [K-A' greater than or equal to 10(8) M-1 at 4 or 20 degreesC], as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg2+ also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(HI89A), increasing the transition temperature (T-m) for unfolding the C-terminal domain by similar to18 degreesC and that for the N-terminal domain by similar to9 degreesC to T-max congruent to 63 degreesC, giving a value of K-D' congruent to 3 muM PEP at 45 degreesC. PEP alone also promotes the dimerization of EI(H189A) but only increases T-m similar to5 degreesC for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated value of K-D' congruent to 0.2 mM for PEP dissociation in the absence of Mg2+ at 45 degreesC. In contrast, the dimerization constant of phospho-EI at 20 degreesC is the same in the absence and presence of 5 mM PEP and 2 MM MgCl2. Thus, the separation of substrate binding effects from those of phosphorylation by studies with the inactive EI(H189A) has shown that intracellular concentrations of PEP and Mg2+ are important determinants of both the conformational stability and dimerization of dephospho-EI.