Figure 1.

a) Nucleotide‐free UmpK (H. sapiens, PDB: 1TEV10). b) Overlay of UmpK in complex with the bi‐substrate inhibitor AP₅U, (red, D. discoideum, PDB: 1UKE11), and in complex with ADP and CMP (orange, D. discoideum, PDB: 2UKD7). c) UmpK in complex with the TSA ADP:AlF_x:CMP (D. discoideum, PDB: 3UKD7). In (a–c) arginine side‐chains are shown in yellow (numbering refers to UmpK from D. discoideum). d) Schematic representation of the reaction catalyzed by UmpK and the states studied here: nucleotide‐free (green), substrate/product‐bound (red), and the transition state (blue). e) AP₅U binding to UmpK monitored by equilibrium displacement of fluorescent MANT‐ADP. Binding of AP₅U (K _D≤1.0 nm) generates a stable substrate/product‐like state. f) Nucleotide exchange kinetics measured by stopped flow. UmpK incubated with UMP and ADP in the presence of 0.5 mm AlF_x (blue), 3 mm MgF_x (grey) or in the absence of any fluoride (red) was mixed rapidly with the competing ligand MANT‐AP₅A‐MANT. Notably, the dissociation of ADP and UMP is 2000‐fold slower in the presence of AlF_x (k _slow≈0.03 s⁻¹) than in the absence of metal fluoride (k _fast≈60 s⁻¹) and the complex with AlF_x is characterized exclusively by slow nucleotide exchange.