Institute of Biomedical Chemistry, 10, Pogodinskaya St., 119121 Moscow, Russia.
Joint Institute for High Temperatures of Russian Academy of Sciences, 13 Bd.2, Izhorskaya St., 125412 Moscow, Russia.
Int J Mol Sci. 2024 Oct 9;25(19):10864. doi: 10.3390/ijms251910864.
Experimental methods of single-molecule enzymology allow scientists to determine physicochemical properties of distinct single molecules of various enzymes and to perform direct monitoring of functioning of enzymes at different steps of their catalytic cycle. The approach based on the use of solid-state nanopores is a promising tool for studying the functioning of single-enzyme molecules. Herein, this approach is employed for monitoring the functioning of cytochrome P450 BM3, which represents a very convenient model of cytochrome P450-containing monooxygenase systems. A nanopore of ~5 nm in diameter has been formed in a 40 nm-thick silicon nitride chip by electron beam drilling (EBD), and a single molecule of the BM3 enzyme has been entrapped in the pore. The functioning of the enzyme molecule has been monitored by recording the time dependence of the ion current through the nanopore during the reaction of laurate hydroxylation. In our experiments, the enzyme molecule has been found to be active for 1500 s. The results of our research can be further used in the development of highly sensitive detectors for single-molecule studies in enzymology.
单分子酶学的实验方法使科学家能够确定各种酶的不同单个分子的物理化学性质，并直接监测酶在其催化循环的不同步骤中的功能。基于使用固态纳米孔的方法是研究单酶分子功能的一种很有前途的工具。在此，该方法用于监测细胞色素 P450 BM3 的功能，它代表了细胞色素 P450 包含的单加氧酶系统的非常方便的模型。通过电子束钻孔 (EBD) 在 40nm 厚的氮化硅芯片中形成了直径约 5nm 的纳米孔，并将 BM3 酶的单个分子困在孔中。通过记录在月桂酸羟化反应过程中通过纳米孔的离子电流的时间依赖性来监测酶分子的功能。在我们的实验中，发现酶分子在 1500s 内保持活性。我们研究的结果可进一步用于开发用于酶学中单分子研究的高灵敏度检测器。
