Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, PO Box 4800, Christchurch, 8140, New Zealand.
MRC-University of Glasgow Centre for Virus Research, Glasgow, G61 1QH, UK.
Eur Biophys J. 2020 Dec;49(8):761-772. doi: 10.1007/s00249-020-01466-5. Epub 2020 Sep 25.
Pyruvate kinase catalyses the final step of the glycolytic pathway in central energy metabolism. The monomeric structure comprises three domains: a catalytic TIM-barrel, a regulatory domain involved in allosteric activation, and a lid domain that encloses the substrates. The lid domain is thought to close over the TIM-barrel domain forming contacts with the substrates to promote catalysis and may be involved in stabilising the activated state when the allosteric activator is bound. However, it remains unknown whether the lid domain is essential for pyruvate kinase catalytic or regulatory function. To address this, we removed the lid domain of Escherichia coli pyruvate kinase type 1 (PK) using protein engineering. Biochemical analyses demonstrate that, despite the absence of key catalytic residues in the lid domain, PK retains a low level of catalytic activity and has a reduced binding affinity for the substrate phosphoenolpyruvate. The enzyme retains allosteric activation, but the regulatory profile of the enzyme is changed relative to the wild-type enzyme. Analytical ultracentrifugation and small-angle X-ray scattering data show that, beyond the loss of the lid domain, the PK structure is not significantly altered and is consistent with the wild-type tetramer that is assembled through interactions at the TIM and regulatory domains. Our results highlight the contribution of the lid domain for facilitating pyruvate kinase catalysis and regulation, which could aid in the development of small molecule inhibitors for pyruvate kinase and related lid-regulated enzymes.
丙酮酸激酶在中心能量代谢中催化糖酵解途径的最后一步。单体结构由三个结构域组成：一个催化 TIM 桶，一个参与变构激活的调节域，以及一个封闭底物的盖子域。盖子域被认为覆盖 TIM 桶结构域，与底物形成接触以促进催化作用，并且当结合变构激活剂时可能参与稳定激活状态。然而，盖子域是否对丙酮酸激酶的催化或调节功能至关重要仍然未知。为了解决这个问题，我们使用蛋白质工程去除了大肠杆菌丙酮酸激酶 1 型（PK）的盖子域。生化分析表明，尽管盖子域中缺乏关键的催化残基，但 PK 仍然保留低水平的催化活性，并且对底物磷酸烯醇丙酮酸的结合亲和力降低。该酶保留变构激活，但酶的调节谱相对于野生型酶发生了变化。分析超速离心和小角 X 射线散射数据表明，除了盖子域的丢失外，PK 结构没有明显改变，并且与通过 TIM 和调节结构域相互作用组装的野生型四聚体一致。我们的结果强调了盖子域对促进丙酮酸激酶催化和调节的贡献，这可能有助于开发丙酮酸激酶和相关盖子调节酶的小分子抑制剂。
