Mechanistic insights into F420-dependent glucose-6-phosphate dehydrogenase using isotope effects and substrate inhibition studies.
Mechanistic insights into F420-dependent glucose-6-phosphate dehydrogenase using isotope effects and substrate inhibition studies.
复制标题
利用同位素效应和底物抑制研究深入了解 F420 依赖性葡萄糖-6-磷酸脱氢酶的机制。
DOI：
10.1016/j.bbapap.2017.08.001
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Johnson-Winters,Kayunta
中科院分区：
文献类型：
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作者：
Oyugi,MercyA;Bashiri,Ghader;Baker,EdwardN;Johnson-Winters,Kayunta
依赖于F420的葡萄糖-6-磷酸脱氢酶(FGD)参与了分枝杆菌磷酸戊糖途径的关键步骤，它催化葡萄糖-6-磷酸(G6P)与F420辅因子之间的反应，生成6-磷酸葡萄糖内酯和还原辅因子F420H2。在这里，我们的目的是通过末端抑制实验以及溶剂和底物的氚同位素效应研究来探索FGD反应的机理。用柠檬酸作为抑制剂进行的末端抑制研究显示，G6P和F420分别以竞争性和非竞争性的抑制模式存在，这表明了底物有序添加的机制，即F420辅因子必须首先与FGD结合，然后才能与G6P结合。对于G6P和F420，溶剂氚同位素效应研究都得到了正常的溶剂动力学同位素效应(SKIE)onkcatandkcat/Km。质子清单数据得到的分馏系数为0.37，这表明造成观测到的天空的单个质子很可能是Glu109捐赠的，并质子化了N1位置的辅助因子。用G6P和G6P-d1对稳定状态下的底物氚同位素效应进行研究，得到两种情况下的Kie均为1.1，而稳态前的Kie onkos为1.4。由于转移到F420的C5上的氢化物是同位素取代的靶标，这些Kie值进一步支持了我们之前的发现，即氢化物转移在FGD反应中可能不是限速的。
F420-dependent glucose-6-phosphate dehydrogenase (FGD) is involved in the committed step of the pentose phosphate pathway within mycobacteria, where it catalyzes the reaction between glucose-6-phosphate (G6P) and the F420cofactor to yield 6-phosphogluconolactone and the reduced cofactor, F420H2. Here, we aim to probe the FGD reaction mechanism using dead-end inhibition experiments, as well as solvent and substrate deuterium isotope effects studies. The dead-end inhibition studies performed using citrate as the inhibitor revealed competitive and uncompetitive inhibition patterns for G6P and F420respectively, thus suggesting a mechanism of ordered addition of substrates in which the F420cofactor must first bind to FGD before G6P binding. The solvent deuterium isotope effects studies yielded normal solvent kinetic isotope effects (SKIE) onkcatandkcat/Kmfor both G6P and F420. The proton inventory data yielded a fractionation factor of 0.37, suggesting that the single proton responsible for the observed SKIE is likely donated by Glu109 and protonates the cofactor at position N1. The steady state substrate deuterium isotope effects studies using G6P and G6P-d1yielded KIE of 1.1 for bothkcatandkcat/Km, while the pre-steady state KIE onkobswas 1.4. Because the hydride transferred to C5 of F420was the one targeted for isotopic substitution, these KIE values provide further evidence to support our previous findings that hydride transfer is likely not rate-limiting in the FGD reaction.