Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles
Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles
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DOI：
10.1073/pnas.1116670109
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发表时间：
2012-05-29
影响因子：
11.1
通讯作者：
Haran, Gilad
中科院分区：
文献类型：
--
作者：
Piwonski, Hubert M.;Goomanovsky, Mila;Haran, Gilad
产物分子对酶的抑制作用是一种重要而普遍的现象。我们描述了一种在单分子水平上研究产物抑制的方法。单个HRP分子被困在表面系留的脂泡中，并探测它们与荧光底物的反应。当底物很容易渗透到囊泡中时，带电的产物(Resufin)会被捕获并在囊泡中积累。令人惊讶的是，当几十个产物分子积累时，单个酶分子被发现停滞不前。大量的酶学实验证实，该酶是被Resufin非竞争性抑制的。单个酶分子的初始反应速度和完全抑制它们所需的产物分子的数量是广泛分布和动态无序的。然而，这两个看似不相关的参数在每个酶分子中都被发现在很长一段时间内相互关联。这些结果表明，作为一种对抗紊乱的方法，酶已经进化出一种方法来关联结构上不同功能位点的波动。
Enzymatic inhibition by product molecules is an important and widespread phenomenon. We describe an approach to study product inhibition at the single-molecule level. Individual HRP molecules are trapped within surface-tethered lipid vesicles, and their reaction with a fluorogenic substrate is probed. While the substrate readily penetrates into the vesicles, the charged product (resorufin) gets trapped and accumulates inside the vesicles. Surprisingly, individual enzyme molecules are found to stall when a few tens of product molecules accumulate. Bulk enzymology experiments verify that the enzyme is noncompetitively inhibited by resorufin. The initial reaction velocity of individual enzyme molecules and the number of product molecules required for their complete inhibition are broadly distributed and dynamically disordered. The two seemingly unrelated parameters, however, are found to be substantially correlated with each other in each enzyme molecule and over long times. These results suggest that, as a way to counter disorder, enzymes have evolved the means to correlate fluctuations at structurally distinct functional sites.