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首页> 外文期刊>Biochemistry >Equilibrium Binding Studies of a Tryptophan-Shifted Mutant of Phosphofructokinase from Bacillus stearothermophilus
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Equilibrium Binding Studies of a Tryptophan-Shifted Mutant of Phosphofructokinase from Bacillus stearothermophilus

机译:嗜热脂肪芽孢杆菌磷酸果糖激酶的色氨酸移位突变体的平衡结合研究

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A tryptophan-shifted mutant of phosphofructokinase (PFK) from Bacillus stearothermophilus has 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 :1:: 0.04 and 90.4 :1:: 3.5 .uM, 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 :1:: 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 MgA TP 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 from E. coli in which the sign of the coupling free energy is established by the sign of the coupling enthalpy.
机译:已经构建了嗜热脂肪芽孢杆菌的磷酸果糖磷酸激酶(PFK)的色氨酸移位突变体。该突变体在功能上类似于野生型,为在平衡条件下检查PFK的变构性质提供了机会。色氨酸移位突变酶W179F / F230W的独特荧光性质已被用于推论在没有催化转换的情况下配体结合和变构扰动的热力学。具体而言,可以使用色氨酸荧光直接观察到磷酸(烯醇)丙酮酸(PEP)和MgADP与突变体PFK的结合,并且这些配体的解离常数已被测得等于2.71:1 :: 0.04和90.4:1: :分别为3.5 .uM。另外,首次评估了变构配体的同向偶联。 PEP以2.9:1 :: 0.3的希尔数协同结合,而MgADP结合不协同。还确定了这些配体与底物6-磷酸果糖(Fru-6-P)之间的平衡偶联,并且在使用野生型PFK的稳态动力学测定条件下观察到的温度变化趋势相同,表明存在结合的MgA TP对变构相互作用几乎没有影响。像野生型PFK一样,突变体的耦合自由能来自在25°C时对焓和熵分量的大量补偿。此外,表示变构效应性质的每个耦合自由能的符号与焓贡献的符号相反,因此归因于相关的熵变化的更大的绝对值。该特征与来自大肠杆菌的同源PFK中的变构反应的热力学基础直接相反,在该PFK中,通过耦合焓的符号建立了耦合自由能的符号。

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