Substrate Effects on the Stability and Dimerization of t

这是一个持续的项目，Mariana Dimitrova为其进行了许多新的测量，包括在PEP、丙酮酸和镁(II)存在下的非活性EI(H189A)的测量。 细菌磷酸烯醇式丙酮酸(PEP)：糖磷酸转移酶系统(PTS)偶联特定糖的磷酸化和跨膜转运。该途径中的第一个蛋白质，酶I(EI)的活性受单体-二聚体平衡的调节，其中由PEP进行的依赖于镁(II)的自动磷酸化需要二聚体。在不同条件下，用pH 7.5、4和20℃的沉降平衡法测定了大肠杆菌PTS以及活性中心His189被Glu或Ala取代的突变体[EI(H189E)或EI(H189A)]的脱磷和磷酸EI的二聚化常数。同时，用差示扫描量热法和本征色氨酸残基荧光的变化监测了这些形式的EI的热去折叠。与去磷EI和EI(H189A)相比，磷酸化的EI和EI(H189E)在20℃时的二聚化常数[logK=6.3，每M单体表示]比去磷EI和EI(H189A)的二聚化常数高10倍，1 mM的PEP和2 mM的镁(II)[LOG K&GT；8在4℃或20℃时]强烈促进二聚反应，就像EI(H189A)不能发生自磷酸化一样。一起，1 mMPEP和2 mM镁(II)还显著稳定和耦合EI(H189A)的C-末端结构域和N-末端结构域的展开-将C-末端结构域展开的转变温度(Tm)提高约18℃，将N-末端结构域的转变温度(Tm)提高约9℃至Tm=63℃，给出45℃时PEP从C-结构域解离的解离常数约0.003 mm，单独PEP还促进EI(H189A)的二聚化，但仅增加C-末端结构域展开的Tm约5℃而不影响N-末端结构域的展开，在45℃下，在没有镁(II)的情况下，PEP的解离常数估计为0.2 mM。相反，5 mM的丙酮酸与0.2-1.0 mM的PEP和2 mM的镁(II)的存在解偶联N-末端和C-末端结构域的展开，并促进单体EI(H189A)，表明细胞内镁(II)、丙酮酸和PEP的浓度控制着磷酸转移酶系统第一步的酶I的活性。因此，通过对野生型EI和非活性EI(H189A)的研究，底物结合效应与磷酸化效应的分离表明，细胞内镁(II)、PEP和丙酮酸的浓度是决定脱磷酸酶I构象稳定性和二聚化的重要决定因素。 此外，底物类似物磷酸丙酸(0。65 mM)在2 mM镁(II)存在下缓慢与野生型酶I结合，并在与1 mM PEP和2 mM镁(II)孵育时缓慢稳定类似EI(H189A)的构象。这一观察结果在获得适合于X射线结构分析的酶I晶体方面具有潜在的用途。

This is a continuing project for which Mariana Dimitrova has made many new measurements including those with the inactive EI(H189A) in the presence of PEP, pyruvate, and Mg(II). The bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) 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 the Mg(II)-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI of the E. coli PTS as well as for mutants in which Glu or Ala is substituted for the active-site His189 [EI(H189E) or EI(H189A), respectively] have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5, 4 and 20 C. Concurrently, thermal unfolding of these forms of EI have 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 [log K = 6.3, expressed per M monomer] compared to those of dephospho-EI and EI(H189A) at 20 C. Dimerization is strongly promoted by 1 mM PEP with 2 mM Mg(II) [log K > 8 at 4 or 20 C], as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg(II) also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(H189A) - increasing the transition temperature (Tm) for unfolding the C-terminal domain by ca. 18 C and that for the N-terminal domain by ca. 9 C to Tm = 63 C, giving a dissociation constant of ca. 0.003 mM for the dissociation of PEP from the C-domain at 45 C. PEP alone also promotes the dimerization of EI(H189A), but only increases Tm ca. 5 C for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated dissociation constant of 0.2 mM for PEP dissociation in the absence of Mg(II) at 45 C. In contrast, the presence of 5 mM pyruvate with 0.2-1.0 mM PEP and 2 mM Mg(II) decouples the unfolding of N- and C-terminal domains and promotes monomeric EI(H189A), indicating that intracellular concentrations of Mg(II), pyruvate, and PEP control the activity of enzyme I in the first step of the phosphotransferase system. Thus, the separation of substrate binding effects from those of phosphorylation by studies with wild-type EI and the inactive EI(H189A) has shown that intracellular concentrations of Mg(II), PEP, and pyruvate are important determinants of both conformational stability and dimerization of dephospho-enzyme I. In addition, the substrate analogue phosphonopyuvate (0. 65 mM) has been found to slowly bind to wild-type enzyme I in the presence of 2 mM Mg(II) and to slowly stabilize a conformation similar to that of EI(H189A) when incubated with 1 mM PEP and 2 mM Mg (II). This observation has a potential use in obtaining crystals of enzyme I suitable for x-ray structural analysis.