Conversion of an amide to a high-energy thioester by Staphylococcus aureus sortase A is powered by variable binding affinity for calcium.
Conversion of an amide to a high-energy thioester by Staphylococcus aureus sortase A is powered by variable binding affinity for calcium.
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金黄色葡萄球菌分选酶 A 将酰胺转化为高能硫酯的过程是通过与钙的不同结合亲和力来实现的。
DOI：
10.1038/s41598-018-34752-6
复制
发表时间：
2018-11-06
影响因子：
4.6
通讯作者：
Su XC
中科院分区：
文献类型：
--
作者：
Wang X;Chen JL;Otting G;Su XC
硫酯是生物学中的关键中间体，通常由能量较低的酰胺前体产生。金黄色葡萄球菌（Staphylococcus aureus sortase A, SrtA）是一种广泛应用于生物技术的肽连接酶。反应分两步进行，第一步是用酶将底物肽的酰胺键转化为硫酯中间体。在这里，我们发现这一步所需的自由能与SrtA的钙结合位点上钙离子的结合亲和力增加了约30倍相匹配，而SrtA的钙结合位点远离硫酯键。这种变构效应的大小突出了钙对SrtA活性的重要性。底物结合时钙结合亲和力的增加，不仅在没有三磷酸核苷酸或任何紧密的非共价酶-底物相互作用的情况下催化形成了富能中间体，而且还伴随着不稳定的硫酯中间体的积累，这使得它在核磁共振（NMR）光谱中可以直接观察到。
Thioesters are key intermediates in biology, which often are generated from less energy-rich amide precursors. Staphylococcus aureus sortase A (SrtA) is an enzyme widely used in biotechnology for peptide ligation. The reaction proceeds in two steps, where the first step involves the conversion of an amide bond of substrate peptide into a thioester intermediate with the enzyme. Here we show that the free energy required for this step is matched by an about 30-fold increase in binding affinity of a calcium ion at the calcium binding site of SrtA, which is remote from the thioester bond. The magnitude of this allosteric effect highlights the importance of calcium for the activity of SrtA. The increase in calcium binding affinity upon binding of substrate not only achieves catalytic formation of an energy-rich intermediate in the absence of nucleotide triphosphates or any tight non-covalent enzyme-substrate interactions, but is also accompanied by accumulation of the labile thioester intermediate, which makes it directly observable in nuclear magnetic resonance (NMR) spectra.
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DOI：
10.1016/bs.apcsb.2017.04.008
发表时间：
2017
影响因子：
--
作者：
Jacobitz AW;Kattke MD;Wereszczynski J;Clubb RT
通讯作者：
Clubb RT
影响因子：
2.7
作者：
Freiburger, Lee;Sonntag, Miriam;Sattler, Michael
通讯作者：
Sattler, Michael
影响因子：
2.7
作者：
Hou, Xiaochen;Wang, Meining;Yang, Cai-Guang
通讯作者：
Yang, Cai-Guang
影响因子：
14.9
作者：
Perler, FB
通讯作者：
Perler, FB
影响因子：
4.8
作者：
Naik, MT;Suree, N;Clubb, RT
通讯作者：
Clubb, RT