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首页> 外文期刊>Journal of Molecular Biology >Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima: Catalytic, spectral and thermodynamic properties
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Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima: Catalytic, spectral and thermodynamic properties

机译：来自嗜热嗜热菌的重组磷酸甘油酸激酶：催化，光谱和热力学性质

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Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima (TmPGK) has been expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity applying heat incubation of the crude extract at 80 degrees C, ion exchange chromatography and gel filtration. The biochemical, catalytic and spectral properties were compared with those of the natural enzyme and found to be identical. As shown by SDS-PAGE, ultracentrifugal analysis and gel filtration chromatography, the enzyme is a 43 kDa monomer. At neutral pH, the guanidinium chloride (GdmCl) and temperature-induced denaturation transitions reveal two-state behaviour with high cooperativity. As taken from the temperature dependence of the free energy of unfolding at zero GdmCl concentration and pH 7, optimum stability is observed at approximately 30 degrees C. The difference in the free energies of stabilization for the enzymes from yeast and Thermotoga amounts to Delta Delta G = 85 kJ/mol. The extrapolated temperatures of cold and heat-denaturation are about - 10 and +85 degrees C. This indicates that the stability profile of TmPGK is shifted to higher free energy values and broadened over a wider temperature range, compared to that observed for PGKs from mesophiles or moderately thermophiles. In order to achieve cold or heat-denaturation, GdmCl concentrations of similar to 1.8 or similar to 0.9 M are required. Due to a kinetic intermediate on the pathway of cold denaturation, equilibration in the transition range takes exceedingly long. (C) 1998 Academic Press. [References: 36]
机译：来自嗜热嗜热菌（Thermotoga maritima）（TmPGK）的重组磷酸甘油酸激酶已在大肠杆菌中表达。通过在80℃下对粗提取物进行热孵育，离子交换色谱和凝胶过滤，将重组酶纯化至均质。将生化，催化和光谱性质与天然酶进行比较，发现它们是相同的。如SDS-PAGE，超速离心分析和凝胶过滤色谱所示，该酶为43 kDa单体。在中性pH下，氯化胍（GdmCl）和温度诱导的变性转变显示出具有高协同性的两态行为。从零GdmCl浓度和pH值为7时解折叠自由能的温度依赖性得出，在大约30摄氏度时观察到最佳稳定性。来自酵母和Thermotoga的酶的稳定化自由能之差为Delta Delta G = 85 kJ / mol。冷和热变性的外推温度分别约为-10和+85摄氏度。这表明，与嗜温菌PGK相比，TmPGK的稳定性曲线转移到更高的自由能值并在更宽的温度范围内变宽。或适度的嗜热菌。为了实现冷变性或热变性，需要的GdmCl浓度必须接近1.8或接近0.9M。由于冷变性途径上的动力学中间体，过渡范围内的平衡需要非常长的时间。 （C）1998年学术出版社。 [参考：36]

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