Equilibrium binding studies of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus.
Equilibrium binding studies of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus.
复制标题
嗜热脂肪芽孢杆菌磷酸果糖激酶色氨酸转移突变体的平衡结合研究。
DOI：
10.1021/bi002448i
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Reinhart,GD
中科院分区：
文献类型：
--
作者：
Riley-Lovingshimer,MR;Reinhart,GD
构建了嗜热脂肪芽孢杆菌磷酸果糖激酶（PFK）的突变株。该突变体在功能上与野生型相似，提供了在平衡条件下检查PFK的变构性质的机会。W179 F/F230 W突变酶的独特荧光性质已被用于推导配体结合的热力学和在没有催化周转的情况下的变构扰动。具体而言，磷酸（烯醇）丙酮酸（PEP）和MgADP与突变体PFK的结合可以使用色氨酸荧光直接观察到，并且这些配体的解离常数已被测量为分别等于2.71 ± 0.04和90.4 ± 3.5 μM。此外，同调耦合的变构配体已被评估为第一次。PEP以2.9 ± 0.3的希尔数协同结合，而MgADP结合不是协同的。这些配体和底物果糖6-磷酸（Fru-6-P）之间的平衡耦合也已确定，并遵循相同的趋势与温度下观察到的稳态动力学测定条件下，使用野生型PFK，这表明结合MgATP的存在下有变构相互作用的影响不大。与野生型PFK一样，突变体的耦合自由能主要来自25 °C下的焓和熵分量的补偿。此外，每个耦合自由能的符号，这表明了变构效应的性质，是相反的焓贡献，因此是由于相关的熵变的绝对值较大。这一特性与弗罗姆的同源PFK的变构反应的热力学基础形成直接对比。其中耦合自由能的符号由耦合焓的符号确定。
A tryptophan-shifted mutant of phosphofructokinase (PFK) fromBacillus stearothermophilushas been constructed. This mutant, which is functionally similar to wild-type, provides the opportunity to examine the allosteric properties of PFK under equilibrium conditions. The unique fluorescence properties of the tryptophan-shifted mutant enzyme, W179F/F230W, have been utilized to deduce the thermodynamics of ligand binding and the allosteric perturbations in the absence of catalytic turnover. Specifically, phospho(enol)pyruvate (PEP) and MgADP binding to the mutant PFK can be directly observed using tryptophan fluorescence, and dissociation constants for these ligands have been measured to be equal to 2.71 ± 0.04 and 90.4 ± 3.5 μM, respectively. In addition, the homotropic couplings for the allosteric ligands have been assessed for the first time. PEP binds cooperatively with a Hill number of 2.9 ± 0.3, while MgADP binding is not cooperative. The equilibrium couplings between these ligands and the substrate fructose 6-phosphate (Fru-6-P) have also been determined and follow the same trends with temperature observed under steady-state kinetic assay conditions using wild-type PFK, indicating that the presence of bound MgATP has little influence on the allosteric interactions. Like wild-type PFK, the coupling free energies for the mutant result from largely compensating enthalpy and entropy components at 25 °C. Furthermore, the sign of each coupling free energy, which signifies the nature of the allosteric effect, is opposite that of the enthalpy contribution and is therefore due to the larger absolute value of the associated entropy change. This characteristic stands in direct contrast to the thermodynamic basis of the allosteric response in the homologous PFK fromE. coliin which the sign of the coupling free energy is established by the sign of the coupling enthalpy.