`In crystallo' substrate binding triggers major domain movements and reveals magnesium as a co-activator of Trypanosoma brucei pyruvate kinase.
`In crystallo' substrate binding triggers major domain movements and reveals magnesium as a co-activator of Trypanosoma brucei pyruvate kinase.
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DOI：
10.1107/s0907444913013875
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发表时间：
2013-09
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影响因子：
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通讯作者：
W. Zhong;H. P. Morgan;I. McNae;P. Michels;L. Fothergill-Gilmore;M. Walkinshaw
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文献类型：
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作者：
W. Zhong;H. P. Morgan;I. McNae;P. Michels;L. Fothergill-Gilmore;M. Walkinshaw
丙酮酸激酶（PYK）的活性位点位于酶的AC核心和对应于结构域B的移动的盖之间。许多PYK结构已经被确定，但第一个"效应"结构和第一个PEP（真正的天然底物），现在报道的酶从布氏锥虫。将PEP浸入具有结合的变构激活剂果糖2，6-二磷酸（F26 BP）和Mg（2+）的酶晶体中，触发"晶体中"的B结构域的实质性23 °旋转，导致部分封闭的活性位点。侧链与Mg（2+）和PEP的相互作用可能解释了电畴运动的机理。此外，很明显，当F26BP存在但PEP不存在时，Mg（2+）占据的位置与活性位点处的两个典型Mg（2+）结合位点不同。第三个位点与活性位点相邻，包含与典型位点1相同的氨基酸侧链，但方向不同。位点3的作用是将Mg（2+）螯合在"引发"位置，从而使酶保持其R-状态构象。以这种方式，Mg（2+）与F26BP合作以确保酶处于对底物具有高亲和力的构象。
The active site of pyruvate kinase (PYK) is located between the AC core of the enzyme and a mobile lid corresponding to domain B. Many PYK structures have already been determined, but the first `effector-only' structure and the first with PEP (the true natural substrate) are now reported for the enzyme from Trypanosoma brucei. PEP soaked into crystals of the enzyme with bound allosteric activator fructose 2,6-bisphosphate (F26BP) and Mg(2+) triggers a substantial 23° rotation of the B domain `in crystallo', resulting in a partially closed active site. The interplay of side chains with Mg(2+) and PEP may explain the mechanism of the domain movement. Furthermore, it is apparent that when F26BP is present but PEP is absent Mg(2+) occupies a position that is distinct from the two canonical Mg(2+)-binding sites at the active site. This third site is adjacent to the active site and involves the same amino-acid side chains as in canonical site 1 but in altered orientations. Site 3 acts to sequester Mg(2+) in a `priming' position such that the enzyme is maintained in its R-state conformation. In this way, Mg(2+) cooperates with F26BP to ensure that the enzyme is in a conformation that has a high affinity for the substrate.