Phosphorylation in the catalytic cleft stabilizes and attracts domains of a phosphohexomutase.
Phosphorylation in the catalytic cleft stabilizes and attracts domains of a phosphohexomutase.
复制标题
催化裂隙中的磷酸化稳定并吸引磷酸己糖变位酶的结构域。
DOI：
10.1016/j.bpj.2014.12.003
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发表时间：
2015
影响因子：
3.4
通讯作者：
VanDoren,StevenR
中科院分区：
文献类型：
--
作者：
Xu,Jia;Lee,Yingying;Beamer,LesaJ;VanDoren,StevenR
磷酸化可以调节酶的活性。 α-D-磷酸己糖变位酶催化裂隙中的磷酰基供体瞬时去磷酸化，而反应中间体在裂隙内完成 180° 重新定向。 52 kDa 细菌磷酸甘露糖变位酶/磷酸葡萄糖变位酶的磷酸化形式不易接触染料或蛋白酶，对化学变性更稳定，并且对 NMR 检测到的跨域 3 核心到并置域 4（每个≥1.3 kcal/mol）以及域 1 和 2 部分的氢交换具有广泛的稳定性。然而，磷酸化会加速域 1 和 2 的特定区域中的氢交换，包括活性位点中的金属结合残基。静电场线揭示了磷酸化 Ser-108 和结构域 4 之间催化裂隙上的吸引力，但当 Ser-108 去磷酸化时则存在排斥力。分子动力学 (MD) 模拟了由于结构域 4 的旋转自由度增强而扩展的去磷酸化形式。MD 轨迹的接触和波动能够正确模拟超过 80% 的因磷酸化而受到氢交换保护或脱保护的位点。磷酸化酶中的静电吸引力导致 1) 结构域 4 靠近结构域 1 和 3； 2）可及性降低； 3) 提高这些领域内的稳定性。磷酸化引起的电致伸缩可能有助于捕获底物，而瞬时去磷酸化时裂口的打开允许中间体旋转。磷酸化对氢交换的长期影响与蛋白激酶的平行报告相一致，表明这些多结构域磷酰基转移酶之间存在概念上的联系。
Phosphorylation can modulate the activities of enzymes. The phosphoryl donor in the catalytic cleft ofα-D-phosphohexomutases is transiently dephosphorylated while the reaction intermediate completes a 180° reorientation within the cleft. The phosphorylated form of 52 kDa bacterial phosphomannomutase/phosphoglucomutase is less accessible to dye or protease, more stable to chemical denaturation, and widely stabilized against NMR-detected hydrogen exchange across the core of domain 3 to juxtaposed domain 4 (each by ≥1.3 kcal/mol) and parts of domains 1 and 2. However, phosphorylation accelerates hydrogen exchange in specific regions of domains 1 and 2, including a metal-binding residue in the active site. Electrostatic field lines reveal attraction across the catalytic cleft between phosphorylated Ser-108 and domain 4, but repulsion when Ser-108 is dephosphorylated. Molecular dynamics (MD) simulated the dephosphorylated form to be expanded due to enhanced rotational freedom of domain 4. The contacts and fluctuations of the MD trajectories enabled correct simulation of more than 80% of sites that undergo either protection or deprotection from hydrogen exchange due to phosphorylation. Electrostatic attraction in the phosphorylated enzyme accounts for 1) domain 4 drawing closer to domains 1 and 3; 2) decreased accessibility; and 3) increased stability within these domains. The electrostriction due to phosphorylation may help capture substrate, whereas the opening of the cleft upon transient dephosphorylation allows rotation of the intermediate. The long-range effects of phosphorylation on hydrogen exchange parallel reports on protein kinases, suggesting a conceptual link among these multidomain, phosphoryl transfer enzymes.