Olson Andrew L, Yao Huili, Herdendorf Timothy J, Miziorko Henry M, Hannongbua Supa, Saparpakorn Patchareenart, Cai Sheng, Sem Daniel S
Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, Wisconsin 53201-1881, USA.
Proteins. 2009 Apr;75(1):127-38. doi: 10.1002/prot.22228.
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 gamma-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 M5P K(d)'s were 6-60 microM in all complexes, consistent with there being little binding synergy. Binding of M5P causes the PMK structure to compress (tau(c) = 13.5 nsec), whereas subsequent binding of Mg-ADP opens the structure up (tau(c) = 15.6 nsec). The overall complex seems to stay very rigid on the psec-nsec timescale with an average NMR order parameter of S(2) approximately 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 gamma-phosphate binding interactions. Indeed, the role of gamma-phosphate interactions may be more subtle than suggested by ATP/ADP comparisons, because the conservative O to NH substitution in the beta-gamma 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.
磷酸甲羟戊酸激酶（PMK）催化甲羟戊酸途径中的一个关键步骤，该途径是人类体内合成类异戊二烯和类固醇的唯一途径。PMK催化将ATP的γ-磷酸基团转移至5-磷酸甲羟戊酸（M5P），形成5-二磷酸甲羟戊酸。鉴于活性位点中四个磷酸基团具有高负电荷密度，使这些磷酸基团靠近以发生反应极具挑战性。因此，形成米氏复合物所需的构象和动力学变化具有重要的机制研究意义。在此，我们利用基于核磁共振的动力学和化学位移扰动测量方法，报告了PMK中底物诱导变化（Mg-ADP、M5P和三元复合物）的特征。在所有复合物中，Mg-ADP和M5P的解离常数（K(d)）为6 - 60微摩尔，这表明几乎不存在结合协同作用。M5P的结合导致PMK结构压缩（旋转相关时间τ(c) = 13.5纳秒），而随后Mg-ADP的结合使结构展开（τ(c) = 15.6纳秒）。在皮秒到纳秒的时间尺度上，整个复合物似乎保持非常刚性，平均核磁共振序参数S(2)约为0.88。数据表明，M5P的添加导致围绕一个铰链区域发生移动，从而允许结构域闭合，这将使M5P结构域靠近ATP以促进催化作用。基于动力学数据，潜在的铰链残基为H55和R93，这是根据它们在PMK同源模型中连接M5P和ATP结构域的延伸区域中的低序参数及其位置确定的。同样，D163可能是与腺苷酸激酶盖子同源的盖子区域的一个铰链残基，该盖子覆盖“沃克-A”催化环。ATP或ADP的结合似乎会引起类似的构象变化；然而，这些观察结果并未表明γ-磷酸基团结合相互作用具有明显作用。实际上，γ-磷酸基团相互作用的作用可能比ATP/ADP比较所暗示的更为微妙，因为ATP的β-γ桥中保守的氧到氮的取代导致亲和力显著降低，并且几乎没有诱导化学位移扰动。就催化残基的定位而言，M5P的结合诱导Gly21（与催化重要的Lys22相邻）刚性化，尽管三元复合物中的交换加宽表明在较慢的时间尺度上仍会发生一些运动。最后，N端的前九个残基高度无序，这表明它们可能是可裂解信号或调节肽序列的一部分。
