Department of Chemistry, University of Texas at Austin , Austin, Texas 78712, United States.
Anal Chem. 2018 Jan 2;90(1):839-846. doi: 10.1021/acs.analchem.7b03591. Epub 2017 Dec 15.
The complex interplay of dynamic protein plasticity and specific side-chain interactions with substrate molecules that allows enzymes to catalyze reactions has yet to be fully unraveled. Top-down ultraviolet photodissociation (UVPD) mass spectrometry is used to track snapshots of conformational fluctuations in the phosphotransferase adenylate kinase (AK) throughout its active reaction cycle by characterization of complexes containing AK and each of four different adenosine phosphate ligands. Variations in efficiencies of UVPD backbone cleavages were consistently observed for three α-helices and the adenosine binding regions for AK complexes representing different steps of the catalytic cycle, implying that these stretches of the protein sample various structural microstates as the enzyme undergoes global open-to-closed transitions. Focusing on the conformational impact of recruiting or releasing the Mg cofactor highlights two loop regions for which fragmentation increases upon UVPD, signaling an increase in loop flexibility as the metal cation disrupts the loop interactions with the substrate ligands. Additionally, the observation of holo ions and variations in UVPD backbone cleavage efficiency at R138 implicate this conserved active site residue in stabilizing the donor phosphoryl group during catalysis. This study showcases the utility of UVPD-MS to provide insight into conformational fluctuations of single residues for active enzymes.
动态蛋白质可塑性与特定侧链相互作用与底物分子的复杂相互作用，使酶能够催化反应，但尚未完全揭示。自上而下的紫外光解（UVPD）质谱法用于通过表征包含 AK 和四种不同腺苷磷酸盐配体的复合物，跟踪磷酸转移酶腺苷酸激酶（AK）在其活性反应循环中构象波动的快照。对于 AK 复合物代表催化循环不同步骤的三个α-螺旋和腺苷结合区域，始终观察到 UVPD 骨干裂解效率的变化，这意味着随着酶经历全局开-闭转变，蛋白质样品的这些区域呈现出各种结构微观状态。关注招募或释放 Mg 辅因子对构象的影响，突出了两个环区，在这些环区中，碎片在 UVPD 后增加，表明随着金属阳离子破坏与底物配体的环相互作用，环的灵活性增加。此外，在 R138 处观察到完整离子和 UVPD 骨干裂解效率的变化，表明该保守的活性位点残基在催化过程中稳定供体磷酸基团。这项研究展示了 UVPD-MS 用于提供对活性酶中单个残基构象波动的深入了解的实用性。
