期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2018
卷号：115
期号：13
页码：3243-3248
DOI：10.1073/pnas.1720448115
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The functional cycle of many proteins involves large-scale motions of domains and subunits. The relation between conformational dynamics and the chemical steps of enzymes remains under debate. Here we show that in the presence of substrates, domain motions of an enzyme can take place on the microsecond time scale, yet exert influence on the much-slower chemical step. We study the domain closure reaction of the enzyme adenylate kinase from Escherichia coli while in action (i.e., under turnover conditions), using single-molecule FRET spectroscopy. We find that substrate binding increases dramatically domain closing and opening times, making them as short as ∼15 and ∼45 µs, respectively. These large-scale conformational dynamics are likely the fastest measured to date, and are ∼100–200 times faster than the enzymatic turnover rate. Some active-site mutants are shown to fully or partially prevent the substrate-induced increase in domain closure times, while at the same time they also reduce enzymatic activity, establishing a clear connection between the two phenomena, despite their disparate time scales. Based on these surprising observations, we propose a paradigm for the mode of action of enzymes, in which numerous cycles of conformational rearrangement are required to find a mutual orientation of substrates that is optimal for the chemical reaction.
