Enzyme I (EI) is a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate. This reaction initiates a five-step phosphorylation cascade in the bacterial phosphotransferase (PTS) transduction pathway. Under physiological conditions, EI exists in an equilibrium between a functional dimer and an inactive monomer. The monomer–dimer equilibrium is a crucial factor regulating EI activity and the phosphorylation state of the overall PTS. Experimental studies of EI’s monomeric state have yet been hampered by the dimer’s high thermodynamic stability, which prevents its characterization by standard structural techniques. In this study, we modified the dimerization domain of EI (EIC) by mutating three amino acids involved in the formation of intersubunit salt bridges. The engineered variant forms an active dimer in solution that can bind and hydrolyze PEP. Using hydrostatic pressure as an additional perturbation, we were then able to study the complete dissociation of the variant from 1 bar to 2.5 kbar in the absence and the presence of EI natural ligands. Backbone residual dipolar couplings collected under high-pressure conditions allowed us to determine the conformational ensemble of the isolated EIC monomeric state in solution. Our calculations reveal that three catalytic loops near the dimerization interface become unstructured upon monomerization, preventing the monomeric enzyme from binding its natural substrate. This study provides an atomic-level characterization of EI’s monomeric state and highlights the role of the catalytic loops as allosteric connectors controlling both the activity and oligomerization of the enzyme.

酶 I (EI) 是一种磷酸转移酶，负责将磷酸烯醇式丙酮酸 (PEP) 转化为丙酮酸。该反应启动了细菌磷酸转移酶 (PTS) 转导途径中的五步磷酸化级联反应。在生理条件下，EI 处于功能二聚体和非活性单体之间的平衡状态。单体-二聚体平衡是调节 EI 活性和整个 PTS 磷酸化状态的关键因素。EI 单体状态的实验研究尚未受到二聚体的高热力学稳定性的阻碍，这阻碍了其通过标准结构技术进行表征。在这项研究中，我们通过突变参与亚基间盐桥形成的三个氨基酸来修饰 EI (EIC) 的二聚化结构域。工程变体在溶液中形成活性二聚体，可以结合和水解 PEP。使用静水压力作为额外的扰动，然后我们能够研究变体在 EI 天然配体不存在和存在的情况下从 1 bar 到 2.5 kbar 的完全解离。在高压条件下收集的骨架残余偶极耦合使我们能够确定溶液中分离的 EIC 单体状态的构象集合。我们的计算表明，二聚化界面附近的三个催化环在单体化时变得非结构化，从而阻止单体酶与其天然底物结合。