Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan.
J Biol Chem. 2011 Jun 24;286(25):22028-34. doi: 10.1074/jbc.M111.240168. Epub 2011 Apr 29.
Both β-galactosidase (GAL) and β-glucuronidase (GUS) are tetrameric enzymes used widely as reporter proteins. However, little is known about the folding and assembly of these enzymes. Although the refolding kinetics of GAL from a denatured enzyme have been reported, it is not known how the kinetics differ when coupled with a protein translation reaction. Elucidating the assembly kinetics of GAL and GUS when coupled with protein translation will illustrate the differences between these two reporter proteins and also the assembly process under conditions more relevant to those in vivo. In this study, we used an in vitro translation/transcription system to synthesize GAL and GUS, measured the time development of the activity and oligomerization state of these enzymes, and determined the rate constants of the monomer to tetramer assembly process. We found that at similar concentrations, GAL assembles into tetramers faster than GUS. The rate constant of monomer to dimer assembly of GAL was 50-fold faster when coupled with protein translation than that of refolding from the denatured state. Furthermore, GAL synthesis was found to lack the rate-limiting step in the assembly process, whereas GUS has two rate-limiting steps: monomer to dimer assembly and dimer to tetramer assembly. The consequence of these differences when used as reporter proteins is discussed.
β-半乳糖苷酶（GAL）和β-葡萄糖醛酸酶（GUS）都是广泛用作报告蛋白的四聚体酶。然而，人们对这些酶的折叠和组装知之甚少。尽管已经报道了从变性酶中复性的 GAL 的重折叠动力学，但尚不清楚当与蛋白质翻译反应偶联时动力学有何不同。阐明与蛋白质翻译偶联时 GAL 和 GUS 的组装动力学将阐明这两种报告蛋白之间的差异，以及更接近体内条件下的组装过程。在这项研究中，我们使用体外翻译/转录系统合成 GAL 和 GUS，测量这些酶的活性和寡聚状态的时间发展，并确定单体到四聚体组装过程的速率常数。我们发现，在相似浓度下，GAL 比 GUS更快地组装成四聚体。当与蛋白质翻译偶联时，GAL 的单体到二聚体组装的速率常数比从变性状态复性时快 50 倍。此外，发现 GAL 合成在组装过程中缺乏限速步骤，而 GUS 有两个限速步骤：单体到二聚体组装和二聚体到四聚体组装。讨论了将它们用作报告蛋白时这些差异的后果。
