Sugahara Michihiro, Ohshima Noriyasu, Ukita Yoko, Sugahara Mitsuaki, Kunishima Naoki
Advanced Protein Crystallography Research Group, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Sayo-gun, Hyogo 679-5148, Japan.
Acta Crystallogr D Biol Crystallogr. 2005 Nov;61(Pt 11):1500-7. doi: 10.1107/S090744490502651X. Epub 2005 Oct 19.
In order to understand the induced fit and the thermostabilization mechanisms of ATP-dependent phosphoenolpyruvate carboxykinase, the crystal structure of the enzyme from the extreme thermophile Thermus thermophilus HB8 (TtPEPCK) was determined and compared with those of orthologues of known structure from two mesophilic organisms. The protomer structures in these orthologues, which exhibit open/closed interdomain conformations, are similar. Isomorphous crystals of unliganded and ATP-bound TtPEPCK were obtained. The asymmetric units of both crystal forms contain two protomers A and B with closed and open conformations, respectively. ATP was only observed in the interdomain cleft of the closed protomer, suggesting that the induced fit of TtPEPCK agrees with the so-called ;conformational selection' mechanism where ligand binding is not essential for domain closure although its binding leads to the stabilization of the closed state. A bound calcium observed in the N-terminal domain of TtPEPCK probably contributes to the thermal stability. A combination of hydrophobic effects, ion pairs and entropic effects might also contribute to the thermostability of TtPEPCK.
为了理解ATP依赖的磷酸烯醇式丙酮酸羧激酶的诱导契合和热稳定机制，我们测定了嗜热栖热菌HB8（TtPEPCK）中该酶的晶体结构，并将其与来自两种嗜温生物的已知结构的直系同源物进行了比较。这些直系同源物中的原体结构呈现出开放/闭合的结构域间构象，它们是相似的。我们获得了未结合配体和结合ATP的TtPEPCK的同晶型晶体。两种晶体形式的不对称单元分别包含两个具有闭合和开放构象的原体A和B。仅在闭合原体的结构域间裂隙中观察到ATP，这表明TtPEPCK的诱导契合符合所谓的“构象选择”机制，即配体结合对于结构域闭合并非必不可少，尽管其结合会导致闭合状态的稳定。在TtPEPCK的N端结构域中观察到的结合钙可能有助于热稳定性。疏水作用、离子对和熵效应的组合也可能有助于TtPEPCK的热稳定性。
