Pelz Benjamin, Žoldák Gabriel, Zeller Fabian, Zacharias Martin, Rief Matthias
Physik Department E22, Technische Universität München, James Franck Strasse 1, 85748 Garching, Germany.
Physik Department T38, Technische Universität München, 85748 Garching, Germany.
Nat Commun. 2016 Feb 24;7:10848. doi: 10.1038/ncomms10848.
Enzymes are molecular machines that bind substrates specifically, provide an adequate chemical environment for catalysis and exchange products rapidly, to ensure fast turnover rates. Direct information about the energetics that drive conformational changes is difficult to obtain. We used subnanometre single-molecule force spectroscopy to study the energetic drive of substrate-dependent lid closing in the enzyme adenylate kinase. Here we show that in the presence of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A), closing and opening of both lids is cooperative and tightly coupled to inhibitor binding. Surprisingly, binding of the substrates ADP and ATP exhibits a much smaller energetic drive towards the fully closed state. Instead, we observe a new dominant energetic minimum with both lids half closed. Our results, combining experiment and molecular dynamics simulations, give detailed mechanical insights into how an enzyme can cope with the seemingly contradictory requirements of rapid substrate exchange and tight closing, to ensure efficient catalysis.
酶是分子机器，能特异性结合底物，为催化作用提供适宜的化学环境并快速交换产物，以确保高周转率。关于驱动构象变化的能量学的直接信息很难获得。我们使用亚纳米级单分子力谱来研究腺苷酸激酶中底物依赖性盖子关闭的能量驱动。在此我们表明，在双底物抑制剂二腺苷五磷酸（AP5A）存在的情况下，两个盖子的关闭和打开是协同的，并且与抑制剂结合紧密耦合。令人惊讶的是，底物ADP和ATP的结合对完全关闭状态的能量驱动要小得多。相反，我们观察到一种新的主要能量最小值，此时两个盖子均半闭。我们结合实验和分子动力学模拟的结果，对酶如何应对快速底物交换和紧密关闭这两个看似矛盾的要求以确保高效催化提供了详细的力学见解。
