Substrate Effects on the Stability and Dimerization of t

这是一个持续的项目，Mariana Dimitrova已经进行了许多新的测量，包括在PEP，丙酮酸和Mg（II）存在下的非活性EI（H189 A）。 细菌磷酸烯醇式丙酮酸（PEP）：糖磷酸转移酶系统（PTS）将特定糖的磷酸化和跨膜转运偶联。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的Mg（II）依赖性自磷酸化需要二聚体。脱磷EI和磷酸EI的二聚化常数。在pH7.5、4和20 ℃的各种条件下，通过沉降平衡法测定了大肠杆菌PTS以及Glu或Ala取代活性位点His 189的突变体[分别为EI（H189 E）或EI（H189 A）]。同时，这些形式的EI的热展开已被监测差示扫描量热法和内在的dahanyl残基荧光的变化。在20 ℃下，与去磷酸化EI和EI（H189 A）相比，磷酸化EI和EI（H189 E）具有10倍增加的二聚化常数[log K = 6.3，以每M单体表示]。如EI（H189 A）所证明的那样，1 mMPEP和2 mMMg（II）强烈促进二聚化[在4或20 ℃时log K > 8]，EI（H189 A）不能进行自磷酸化。同时，1 mM PEP和2 mM Mg（II）也显著稳定和偶联EI（H189 A）的C-和N-末端结构域的解折叠-使解折叠C-末端结构域的转变温度（Tm）增加约100 ℃。18 C和N-末端结构域由ca. 9 ℃至Tm = 63 ℃，得到解离常数约为100。0.003mM，用于PEP在45 ℃从C-结构域解离。单独的PEP也促进EI（H189 A）的二聚化，但仅增加Tm约100。5 ℃时C-末端结构域解折叠，而不影响N-末端结构域解折叠，在45 ℃下不存在Mg（II）时PEP解离的估计解离常数为0.2 mM。相比之下，5 mM丙酮酸与0.2-1.0 mM PEP和2 mM Mg（II）的存在使N-和C-末端结构域的展开更加复杂，并促进单体EI（H189 A），表明Mg（II）、丙酮酸和PEP的细胞内浓度控制磷酸转移酶系统第一步中酶I的活性。因此，通过对野生型EI和失活EI（H189 A）的研究，将底物结合效应与磷酸化效应分开，表明Mg（II）、PEP和丙酮酸盐的细胞内浓度是脱磷酸酶I构象稳定性和二聚化的重要决定因素。 此外，底物类似物膦酰丙酮酸酯（0. 65 mM）在2 mM Mg（II）存在下缓慢结合野生型酶I，并且当与1 mM PEP和2 mM Mg（II）孵育时缓慢稳定类似于EI（H189 A）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晶体中具有潜在的用途。

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.