Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel.
Proc Natl Acad Sci U S A. 2012 May 29;109(22):E1437-43. doi: 10.1073/pnas.1116670109. Epub 2012 May 4.
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.
产物分子的酶抑制作用是一种重要且广泛存在的现象。我们描述了一种在单分子水平上研究产物抑制的方法。将 HRP 分子单个捕获在表面连接的脂质体中，并探测它们与荧光底物的反应。虽然底物很容易渗透到囊泡中，但带电荷的产物（试卤灵）被困在囊泡内并积累。令人惊讶的是，当积累数十个产物分子时，发现单个酶分子会停滞。批量酶学实验验证了酶受到试卤灵的非竞争性抑制。单个酶分子的初始反应速度和完全抑制所需的产物分子数量广泛分布且动态无序。然而，这两个看似不相关的参数在每个酶分子中和长时间内都被发现彼此之间存在实质性关联。这些结果表明，作为应对无序的一种方式，酶已经进化出了在结构上不同的功能位点之间相关波动的手段。
