Delineation of alternative conformational states in Escherichia coli peptide deformylase via thermodynamic studies for the binding of actinonin.
Delineation of alternative conformational states in Escherichia coli peptide deformylase via thermodynamic studies for the binding of actinonin.
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DOI：
10.1021/bi8019542
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发表时间：
2009-02-24
期刊：
影响因子：
2.9
通讯作者：
Srivastava, D. K.
中科院分区：
文献类型：
--
作者：
Berg, Alexander K.;Srivastava, D. K.
我们通过等温滴定微量热法研究了一种天然存在的抗生素actionin与Ni2+重组形式的大肠杆菌肽去甲酰基酶（PDFEc）的结合。结合数据符合放热相和吸热相，其量级分别为ΔG°、ΔH°和TΔS°，分别为−12、−2.7和9.3、−8.7、3.9和12.6 kcal/mol。显然，虽然两相都以有利的熵变为主，但放热相比吸热相有6.7 kcal/mol的焓优势。我们观察到，从PDFEc中去除结合的Ni2+消除了放热相，但不影响吸热相，但在加入Zn2+后又恢复了吸热相。结合金属分析数据，我们提出重组形式的PDFEc在与actionin相互作用时以两种稳定的构象状态表达，产生明显不同的ITC谱（即放热和吸热）。在酶热展开过程中观察到的两个不同且独立的转变进一步支持了PDFEc的两种构象态的存在。此外，热力学数据表明，pdfec - actionin复合物的形成导致一个H+从酶相转移到pH为6.3的体溶剂中。放热和吸热相均产生高度负的ΔCp°值，但对pdfec - actionin复合物的形成没有明显的焓-熵补偿作用。鉴于酶的已知结构特征，提出了PDFEc的不同构象状态是由酶位点的金属连接调节的论点。
We investigated the binding of a naturally occurring antibiotic, actinonin, to the Ni2+ reconstituted recombinant form of E. coli peptide deformylase (PDFEc) via isothermal titration microcalorimetry. The binding data conformed to both exothermic and endothermic phases with magnitudes of ΔG°, ΔH°, and TΔS° being equal to −12, −2.7, and 9.3, and −8.7, 3.9, and 12.6 kcal/mol, respectively. Evidently, although both phases are dominated by favorable entropic changes, the exothermic phase has about 6.7 kcal/mol enthalpic advantage over the endothermic phase. We observed that the removal of bound Ni2+ from PDFEc abolished the exothermic phase without affecting the endothermic phase, but it was regained upon addition of Zn2+. In conjunction with metal analysis data, we propose that the recombinant form of PDFEc is expressed in two stable conformational states that yield markedly distinct ITC profiles (i.e., exothermic versus endothermic) upon interaction with actinonin. The existence of two conformational states of PDFEc is further supported by the observation of two distinct and independent transitions during the thermal unfolding of the enzyme. In addition, the thermodynamic data reveals that the formation of the PDFEc-actinonin complex results in the transfer of one H+ from the enzyme phase to the bulk solvent at pH 6.3. Both exothermic and endothermic phases produce highly negative ΔCp° values, but there is no apparent enthalpy-entropy compensation effect upon formation of the PDFEc-actinonin complex. In view of the known structural features of the enzyme, arguments are presented that the alternative conformational states of PDFEc are modulated by the metal ligation at the enzyme site.
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