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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalvtic cvcle of human P-glycoprotein ytic cycle of human P-glycoprotein
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Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalvtic cvcle of human P-glycoprotein ytic cycle of human P-glycoprotein

机译：在人类P-糖蛋白的空洞循环的单个周转期间需要在两个不同的步骤进行ATP水解的证据

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摘要

P-glycoprotein (Pgp) is an ATP-dependent hydrophobic natural product anticancer drug efflux pump whose overexpression con- fers multidrug resistance to tumor cells. The work reported here deals with the elucidation of the energy requirement for substrate interaction with Pgp during the catalytic cycle. We show that the Kd (412 nM) of the substrate analogue [125l]iodoarylazidoprazoin for Pgp is not altered by the presence of the nonhydrolyzable nucleotide 5'-adenylylimididiphosphate and vanadate (Kd = 403 nM). Though binding of nucleotide per se does not affect interac- tions with the substrate, ATP hydrolysis results in a dramatic conformational change where the affinity of [125I]iodoarylazido- prazoin for Pgp trapped in transition-state conformation (Pgp.ADP.vanadate) is reduced >30-fold. To transform Pgp from this intermediate state of low affinity for substrate to the next catalytic cycle, i.e., a conformation that binds substrate with high affinity. requires conditions that permit ATP hydrolysis. Addition- alloy. there is an inverse correlation (R~2 = 0.96) between 8AzidoADP (or ADP) release and the recovery of Substrate binding. These results suggest that the release of nucleotide is necessary for reactivation but not sufficient. The hydrolysis of additional mole- cule(s) of ATP (or 8AzidoATP) is obligatory for the catalytic cycle to advance to completion. These data are consistent with the ob- served stoichiometry of two ATP molecules hydrolyzed for the transport of every substrate molecule. Our data demonstrate two distinct roles for ATP hydrolysis in a single turnover of the catalytic cyde of Pgp, one in the transport of substrate and the other in effecting conformational changes to reset the pump for the next catalytic cycle.
机译：P-糖蛋白（Pgp）是一种ATP依赖性疏水性天然产物抗癌药物外排泵，其过表达赋予对肿瘤细胞多药耐药性。此处报道的工作阐明了催化循环中底物与Pgp相互作用所需的能量。我们显示，Pgp的底物类似物[125l] iodoarylazidoprazoin的Kd（412 nM）不会因不可水解核苷酸5'-adenylylimididiphosphate和钒酸盐（Kd = 403 nM）的存在而改变。尽管核苷酸本身的结合不会影响与底物的相互作用，但ATP水解会导致构象发生显着变化，其中[125I]碘代芳基叠氮吡嗪对被困在过渡态构象中的Pgp的亲和力为（Pgp.ADP.vanadate）。减少> 30倍。将Pgp从对底物的低亲和力的中间状态转化为下一个催化循环，即以高亲和力结合底物的构象。需要允许ATP水解的条件。附加合金。 8AzidoADP（或ADP）的释放与底物结合的恢复之间存在负相关（R〜2 = 0.96）。这些结果表明核苷酸的释放对于重新激活是必需的，但还不够。 ATP（或8AzidoATP）的其他分子的水解对于催化循环必不可少。这些数据与为每个底物分子的运输而水解的两个ATP分子的化学计量一致。我们的数据表明，ATP在Pgp催化环的单个循环中水解有两个不同的作用，一个在底物的运输中，另一个在影响构象变化以重置泵以进行下一个催化循环中。

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