Substrate induced structural and dynamics changes in human phosphomevalonate kinase and implications for mechanism.
Substrate induced structural and dynamics changes in human phosphomevalonate kinase and implications for mechanism.
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底物诱导人磷酸甲羟戊酸激酶的结构和动力学变化及其机制的影响。
DOI：
10.1002/prot.22228
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发表时间：
2009
期刊：
影响因子：
2.9
通讯作者：
Sem,DanielS
中科院分区：
文献类型：
--
作者：
Olson,AndrewL;Yao,Huili;Herdendorf,TimothyJ;Miziorko,HenryM;Hannongbua,Supa;Saparpakorn,Patchareenart;Cai,Sheng;Sem,DanielS
磷酸甲羟戊酸激酶（PMK）催化甲羟戊酸途径中的重要步骤，甲羟戊酸途径是人类合成类异戊二烯和类固醇的唯一途径。PMK催化ATP的γ-磷酸转移至甲羟戊酸5-磷酸（M5 P），形成甲羟戊酸5-二磷酸。考虑到活性位点中四个磷酸基团上的高负电荷密度，使这些磷酸基团接近反应尤其具有挑战性。因此，形成米氏复合物所需的构象和动力学变化具有机理意义。在本文中，我们使用基于NMR的动力学和化学位移微扰测量报告了PMK中底物诱导变化（Mg-ADP，M5 P和三元复合物）的表征。Mg-ADP和M5 PKd均为6-60 μ π ι η，与几乎没有结合协同作用一致。M5 P的结合导致PMK结构压缩（τc= 13.5 nsec），而随后的Mg-ADP结合打开了结构（τc= 15.6 nsec）。整个复合物似乎在皮秒-纳秒的时间尺度上保持非常刚性，平均NMR序参数为S2 = 0.88。数据与M5 P的添加一致，M5 P的添加引起铰链区周围的移动以允许结构域闭合，这将使M5 P结构域接近ATP以允许催化。动力学数据基于它们的低阶参数和它们在PMK同源性模型中连接M5 P和ATP结构域的延伸区域中的位置，将潜在的铰链残基鉴定为H55和R93。同样，D163可以是与腺苷酸激酶盖同源的盖区的铰链残基，覆盖“步行者-A”催化环。ATP或ADP的结合似乎引起类似的构象变化;然而，这些观察结果并不表明γ-磷酸结合相互作用的明显作用。事实上，γ-磷酸相互作用的作用可能比ATP/ADP比较所表明的更微妙，因为ATP的β-γ桥中保守的O到NH取代导致亲和力急剧下降，并引起很少的化学位移扰动。在定位的催化残基，结合M5 P诱导硬化的Gly 21（相邻的催化重要的Lys 22），虽然在三元复合物中的交换加宽表明一些运动在一个较慢的时间尺度上仍然发生。最后，N末端的前9个残基是高度无序的，表明它们可能是可切割的信号或调节肽序列的一部分。Proteins 2009.© 2008 Wiley利斯公司
Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, which is the only pathway for synthesis of isoprenoids and steroids in humans. PMK catalyzes transfer of the γ‐phosphate of ATP to mevalonate 5‐phosphate (M5P) to form mevalonate 5‐diphosphate. Bringing these phosphate groups in proximity to react is especially challenging, given the high negative charge density on the four phosphate groups in the active site. As such, conformational and dynamics changes needed to form the Michaelis complex are of mechanistic interest. Herein, we report the characterization of substrate induced changes (Mg‐ADP, M5P, and the ternary complex) in PMK using NMR‐based dynamics and chemical shift perturbation measurements. Mg‐ADP and M5PKd's were 6–60 μMin all complexes, consistent with there being little binding synergy. Binding of M5P causes the PMK structure to compress (τc= 13.5 nsec), whereas subsequent binding of Mg‐ADP opens the structure up (τc= 15.6 nsec). The overall complex seems to stay very rigid on the psec‐nsec timescale with an average NMR order parameter ofS2∼0.88. Data are consistent with addition of M5P causing movement around a hinge region to permit domain closure, which would bring the M5P domain close to ATP to permit catalysis. Dynamics data identify potential hinge residues as H55 and R93, based on their low order parameters and their location in extended regions that connect the M5P and ATP domains in the PMK homology model. Likewise, D163 may be a hinge residue for the lid region that is homologous to the adenylate kinase lid, covering the “Walker‐A” catalytic loop. Binding of ATP or ADP appears to cause similar conformational changes; however, these observations do not indicate an obvious role for γ‐phosphate binding interactions. Indeed, the role of γ‐phosphate interactions may be more subtle than suggested by ATP/ADP comparisons, because the conservative O to NH substitution in the β‐γ bridge of ATP causes a dramatic decrease in affinity and induces few chemical shift perturbations. In terms of positioning of catalytic residues, binding of M5P induces a rigidification of Gly21 (adjacent to the catalytically important Lys22), although exchange broadening in the ternary complex suggests some motion on a slower timescale does still occur. Finally, the first nine residues of the N‐terminus are highly disordered, suggesting that they may be part of a cleavable signal or regulatory peptide sequence. Proteins 2009. © 2008 Wiley‐Liss, Inc.