Harvard Medical School, Boston, Massachusetts; Brigham and Women's Hospital, Department of Pathology, Boston, Massachusetts; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts.
Harvard Medical School, Boston, Massachusetts; Brigham and Women's Hospital, Department of Pathology, Boston, Massachusetts; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts; Department of Chemistry, University of Toronto, Mississauga, Ontario, Canada; Department of Chemical and Physical Sciences, University of Toronto, Mississauga, Ontario, Canada.
Biophys J. 2022 Jun 7;121(11):2027-2034. doi: 10.1016/j.bpj.2022.05.005. Epub 2022 May 7.
Single-molecule-enzymology (SME) methods have enabled the observation of heterogeneous catalytic activities within a single enzyme population. Heterogeneous activity is hypothesized to originate from conformational changes in the enzyme that result from changes in the local environment leading to catalytically active substates. Here, we use SME to investigate the mechanisms of heterogeneous activity exhibited by tissue nonspecific alkaline phosphatase (TNSALP), which reveals two subpopulations with different catalytic activities. We show the effect of pH and temperature on the distribution of TNSALP activity and confirm the presence of two subpopulations attributed to half- and fully active TNSALP substates. We provide mechanistic insight about protein structure using molecular dynamic simulations and show pH- and temperature-dependent conformational transitions that corroborate experimentally observed changes in TNSALP activity. These results show the utility of SME to understand heterogeneous enzyme activity and demonstrate a simple approach using pH and temperature to tune catalytic activity within an enzyme population.
单分子酶学（SME）方法使人们能够在单个酶群体中观察到异质催化活性。异质活性假设源自于酶的构象变化，这些变化是由局部环境的变化引起的，导致具有催化活性的亚基。在这里，我们使用 SME 来研究组织非特异性碱性磷酸酶（TNSALP）表现出的异质活性的机制，这揭示了具有不同催化活性的两个亚群。我们展示了 pH 值和温度对 TNSALP 活性分布的影响，并证实了存在两种亚群，归因于半活性和全活性 TNSALP 亚基。我们使用分子动力学模拟提供了关于蛋白质结构的机制见解，并展示了 pH 和温度依赖性构象转变，这些转变与实验观察到的 TNSALP 活性变化相符。这些结果表明 SME 可用于理解异质酶活性，并展示了一种简单的方法，使用 pH 值和温度来调节酶群体中的催化活性。
