In crystallo thermodynamic analysis of conformational change of the topaquinone cofactor in bacterial copper amine oxidase
In crystallo thermodynamic analysis of conformational change of the topaquinone cofactor in bacterial copper amine oxidase
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DOI：
10.1073/pnas.1811837116
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发表时间：
2019-01-02
影响因子：
11.1
通讯作者：
Okajima, Toshihide
中科院分区：
文献类型：
--
作者：
Murakawa, Takeshi;Baba, Seiki;Okajima, Toshihide
在铜胺氧化酶的催化反应中，蛋白质衍生的氧化还原辅因子托帕醌（TPQ）被胺底物还原为氨基甲苯二酚形式（TPQ（amr）），其与半醌自由基（TPQ（Sq））平衡。从TPQ（amr）到TPQ（sq）的转变是一个吸热过程，伴随着辅因子的显著构象变化。我们采用湿空气和gluecoating（HAG）的方法来捕获TPQ（amr）和TPQ（Sq）的非低温冷却的晶体中的酶从节杆菌球形的平衡混合物，并发现平衡移动更多的TPQ（sq）在晶体中比在溶液中。热力学分析的温度依赖的平衡还表明，过渡到TPQ（平方）是熵驱动的晶体和溶液中，给出的热力学参数，导致实验测定的晶体包装效果。此外，我们表明，产品醛的结合的疏水口袋中的活性位点产生各种平衡状态之间的两种形式的产品希夫碱，TPQ（amr），和TPQ（平方），在pH值依赖的方式。温度控制的HAG方法提供了一种用于蛋白质晶体中发生的构象变化的热力学分析的技术，所述蛋白质晶体很难被常规的低温X射线晶体学仔细检查。
In the catalytic reaction of copper amine oxidase, the protein-derived redox cofactor topaquinone (TPQ) is reduced by an amine substrate to an aminoresorcinol form (TPQ(amr)), which is in equilibrium with a semiquinone radical (TPQ(Sq)). The transition from TPQ(amr) to TPQ(sq) is an endothermic process, accompanied by a significant conformational change of the cofactor. We employed the humid air and gluecoating (HAG) method to capture the equilibrium mixture of TPQ(amr) and TPQ(Sq) in noncryocooled crystals of the enzyme from Arthrobacter globiformis and found that the equilibrium shifts more toward TPQ(sq) in crystals than in solution. Thermodynamic analyses of the temperature-dependent equilibrium also revealed that the transition to TPQ(sq) is entropy-driven both in crystals and in solution, giving the thermodynamic parameters that led to experimental determination of the crystal packing effect. Furthermore, we demonstrate that the binding of product aldehyde to the hydrophobic pocket in the active site produces various equilibrium states among two forms of the product Schiff-base, TPQ(amr), and TPQ(sq), in a pH-dependent manner. The temperature-controlled HAG method provides a technique for thermodynamic analysis of conformational changes occurring in protein crystals that are hardly scrutinized by conventional cryogenic X-ray crystallography.