Crystal structures of Escherichia coli glycerol kinase variant S58→W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion
Crystal structures of Escherichia coli glycerol kinase variant S58→W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion
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DOI：
10.1021/bi982460z
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发表时间：
1999-03-23
期刊：
影响因子：
2.9
通讯作者：
Remington, SJ
中科院分区：
文献类型：
--
作者：
Bystrom, CE;Pettigrew, DW;Remington, SJ
大肠杆菌甘油激酶 (GK) 对 ATP 表现出“半位点”反应性，并通过果糖 1,6-二磷酸 (FBP) 进行变构调节，已证明 FBP 可以促进二聚体-四聚体组装并仅抑制四聚体。为了探究四聚体组装的作用，设计了一种突变 (Ser58-->Trp)，以在空间上阻断 FBP 附近二聚体-二聚体界面的形成结合位点 [Ormo, M., Bystrom, C., and Remington, S. J, (1998) Biochemistry 37, 16565-16572] 该取代并没有显着改变米氏常数或改变第二个效应子磷酸载体蛋白 IIA(Glc) 的变构调节；然而，它消除了与不同的不可转移 ATP 类似物和甘油复合物中的 FBP 抑制。揭示了一种不对称二聚体，其中一个亚基采用开放构象，另一个亚基采用先前确定的结构中发现的闭合构象。构象差异是由 N 端结构域相对于 C 端结构域的类似 6.0 度刚体旋转产生的，与在两个亚基中以非生产性构象结合的己糖激酶和肌动蛋白类似。 β,γ-二氟亚甲基腺苷 5'-三磷酸 (AMP-PCF2P) 是 GK 的有效抑制剂，在闭合亚基中非生产性结合，在开放亚基中以假定的生产构象结合，其中 γ-磷酸用于在线转移至甘油。这种不对称性与“半位点”反应性一致，表明 FBP 对 GK 的抑制是由于结构域运动的限制。
Escherichia coli glycerol kinase (GK displays "half-of-the-sites'' reactivity toward ATP and allosteric regulation by fructose 1,6-bisphosphate (FBP, which has been shown to promote dimer-tetramer assembly and to inhibit only tetramers. To probe the role of tetramer assembly, a mutation (Ser58-->Trp) was designed to sterically block formation of the dimer-dimer interface near the FBP binding site [Ormo, M., Bystrom, C., and Remington, S. J, (1998) Biochemistry 37, 16565-16572]. The substitution did not substantially change the Michaelis constants or alter allosteric regulation of GK by a second effector, the phosphocarrier protein IIA(Glc); however, it eliminated FBP inhibition. Crystal structures of GK in complex with different nontransferable ATP analogues and glycerol revealed an asymmetric dimer with one subunit adopting an open conformation and the other adopting the closed conformation found in previously determined structures. The conformational difference is produced by a similar to 6.0 degrees rigid-body rotation of the N-terminal domain with respect to the C-terminal domain, similar to that observed for hexokinase and actin, members of the same ATPase superfamily. Two of the ATP analogues bound in nonproductive conformations in both subunits, However, beta,gamma-difluoromethyleneadenosine 5'-triphosphate (AMP-PCF2P), a potent inhibitor of GK, bound nonproductively in the closed subunit and in a putative productive conformation in the open subunit, with the gamma-phosphate placed for in-line transfer to glycerol, This asymmetry is consistent with "half-of-the-sites" reactivity and suggests that the inhibition of GK by FBP is due to restriction of domain motion.