Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
Protein Sci. 2021 Aug;30(8):1628-1639. doi: 10.1002/pro.4102. Epub 2021 Jun 9.
Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully-active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, k , and the rate constant of productive binding, k , of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, implying that inactive monomer units of ALP are deficient in correct disulfide bond formation and zinc ion coordination. These findings provide basis for further study on molecular mechanism and biogenesis of ALP, and also offer the way to prepare homogenous and active populations of ALP for highly quantitative and sensitive bioassays with ALP.
碱性磷酸酶（ALP）是一种同二聚体酶，已广泛应用于各种生物测定法中，作为疾病标志物和酶探针。数字生物测定法的最新进展彻底改变了基于 ALP 的诊断测定法，如数字 ELISA 的快速发展以及单分子 ALP 同型异构酶的新兴多重分析。然而，在 ALP 分子中发现的固有异质性阻碍了基于 ALP 的定量数字生物测定法。本研究旨在定量分析来自大肠杆菌的单分子 ALP 的活性，并揭示 ALP 的催化活性存在静态异质性，存在两个不同的群体：半活性和完全活性部分。通过连续缓冲交换的数字测定揭示了 ALP 的单分子米氏动力学；半活性分子的催化周转率 k 和完全活性分子的有效结合速率常数 k 的值减半。这些发现表明，半活性 ALP 分子是由无活性和活性单体单元组成的异质二聚体，而完全活性的 ALP 分子由两个活性单元组成。在来自牛肠或虾的其他来源的 ALP 中也观察到了静态异质性，表明这些发现可以在物种间推广。具有二硫键增强剂和尖峰锌离子的无细胞表达 ALP 导致 ALP 的完全活性的同质群体，这表明 ALP 的无活性单体单元在正确的二硫键形成和锌离子配位方面存在缺陷。这些发现为进一步研究 ALP 的分子机制和生物发生提供了基础，也为制备同质和活性的 ALP 群体提供了方法，用于具有 ALP 的高度定量和敏感的生物测定。
