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^TIM+Reg) using protein engineering. Biochemical analyses demonstrate that, despite the absence of key catalytic residues in the lid domain, PK^TIM+Reg 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^TIM+Reg 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.

丙酮酸激酶催化中枢能量代谢中糖酵解途径的最后一步。单体结构包括三个域：催化TIM桶，参与变构活化的调节域和包围底物的盖域。人们认为盖结构域在TIM-桶结构域上闭合，从而与底物形成接触以促进催化作用，并且当结合别构活化剂时，可能参与稳定活化状态。然而，仍然不清楚盖结构域对于丙酮酸激酶催化或调节功能是否必需。为了解决这个问题，我们删除了1型大肠杆菌丙酮酸激酶（PK ^{TIM + Reg}）使用蛋白质工程。生化分析表明，尽管在盖域中不存在关键的催化残基，但PK ^{TIM + Reg}保留了低水平的催化活性，并降低了与底物磷酸烯醇丙酮酸的结合亲和力。该酶保留了变构活化，但是该酶的调节特性相对于野生型酶有所改变。分析性超离心和小角X射线散射数据表明，除了盖区的损失外，PK ^{TIM + Reg}其结构没有明显改变，并且与通过TIM和调控域相互作用而组装的野生型四聚体一致。我们的研究结果突出了盖子结构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。