Multiple Ligand-Bound States of a Phosphohexomutase Revealed by Principal Component Analysis of NMR Peak Shifts.
Multiple Ligand-Bound States of a Phosphohexomutase Revealed by Principal Component Analysis of NMR Peak Shifts.
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DOI：
10.1038/s41598-017-05557-w
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发表时间：
2017-07-13
影响因子：
4.6
通讯作者：
Van Doren SR
中科院分区：
文献类型：
--
作者：
Xu J;Sarma AVS;Wei Y;Beamer LJ;Van Doren SR
酶在其催化循环中对多种构象进行采样。核磁共振的化学位移对溶液中的构象变化和系综非常敏感。磷酸甘露糖变位酶/磷酸葡萄糖变位酶(PMM/PGM)是一种普遍存在的四区酶，催化磷酸已糖底物间的磷酸转移。我们比较了酶在催化周期中的访问状态。用核磁共振检测的滴定方法评价假单胞菌PMM/PGM对磷糖底物和抑制剂的集体反应。根据主成分分析(PCA)得到的结合等温线估计亲和力。通过对滴定结果的主成分分析比较，发现了磷酸糖酶之间的关系。协调化学位移行为(简明)分析提供了三种配体结合的PMM/PGM状态的新区分，该状态含有抑制活性的突变。酶的磷酸化和磷酸糖的结合似乎驱动开放的去磷酸化的酶到游离的磷酸化状态，并继续向配体关闭的状态。结构域4似乎是底物和抑制剂结合的集体反应的中心。氢交换表明，底物类似物的结合将结构域的折叠稳定性提高到统一的水平，建立了全球统一的结构。核磁共振谱的简明和主成分分析发现了一种研究得很好的酶的新状态，并似乎准备好区分其他酶和配体结合状态。
Enzymes sample multiple conformations during their catalytic cycles. Chemical shifts from Nuclear Magnetic Resonance (NMR) are hypersensitive to conformational changes and ensembles in solution. Phosphomannomutase/phosphoglucomutase (PMM/PGM) is a ubiquitous four-domain enzyme that catalyzes phosphoryl transfer across phosphohexose substrates. We compared states the enzyme visits during its catalytic cycle. Collective responses of Pseudomonas PMM/PGM to phosphosugar substrates and inhibitor were assessed using NMR-detected titrations. Affinities were estimated from binding isotherms obtained by principal component analysis (PCA). Relationships among phosphosugar-enzyme associations emerge from PCA comparisons of the titrations. COordiNated Chemical Shifts bEhavior (CONCISE) analysis provides novel discrimination of three ligand-bound states of PMM/PGM harboring a mutation that suppresses activity. Enzyme phosphorylation and phosphosugar binding appear to drive the open dephosphorylated enzyme to the free phosphorylated state, and on toward ligand-closed states. Domain 4 appears central to collective responses to substrate and inhibitor binding. Hydrogen exchange reveals that binding of a substrate analogue enhances folding stability of the domains to a uniform level, establishing a globally unified structure. CONCISE and PCA of NMR spectra have discovered novel states of a well-studied enzyme and appear ready to discriminate other enzyme and ligand binding states.
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