National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
PLoS One. 2012;7(7):e40487. doi: 10.1371/journal.pone.0040487. Epub 2012 Jul 11.
Interferon-γ induced human guanylate binding protein-1(hGBP1) belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1.
干扰素-γ诱导的人鸟苷酸结合蛋白-1（hGBP1）属于与动力蛋白相关的大型 GTP 酶家族。与所有其他 GTP 酶不同，hGBP1 将 GTP 水解为 GDP 和 GMP 的混合物，在 37°C 时 GMP 是主要产物，但在 15°C 时 GDP 成为主要产物。hGBP1 的水解反应被认为涉及多个催化步骤。为了研究温度对产物形成和 hGBP1 不同催化复合物的影响，我们进行了依赖于温度的 GTPase 测定、突变分析、化学和热变性研究。有趣的是，GDP 和 GMP 的 Arrhenius 图均表现出非线性行为，表明 GTP 结合酶复合物的产物形成至少涉及多个步骤。GDP 形成的负活化能和外源性 GDP 的 GTPase 测定一起表明，GDP 的形成是通过 GDP 结合酶二聚体可逆解离为单体，然后进一步可逆解离生成产物。不同催化复合物的变性研究表明，与其他复合物不同，游离态 GDP 结合 hGBP1 的自由能在较低温度下显著降低。GDP 的形成取决于 GDP 结合酶复合物的自由能。与高温相比，低温下该复合物的自由能降低是 GDP 形成率在低温下更高的原因。热变性研究还表明，与单体相比，GTP 结合酶二聚体的自由能差异在产物形成中起着至关重要的作用；较高的稳定性有利于 GMP，但较低的稳定性有利于 GDP。因此，本研究首次从热力学角度深入了解了温度对 hGBP1 产物形成的影响。
