Eliminating Competition: Characterizing and Eliminating Competitive Binding at Separate Sites between DAHP Synthase’s Essential Metal Ion and the Inhibitor DAHP Oxime

3-Deoxy-d- arabinoheptulosonate 7-phosphate (DAHP) oxime is a transition state mimic inhibitor of bacterial DAHP synthase, with K _i = 1.5 μM and a residence time of t_R = 83 min. Unexpectedly, DAHP oxime inhibition is competitive with respect to the essential metal ion, Mn²⁺, even though the inhibitor and metal ion do not occupy the same physical space in the active site. This is problematic because DAHP synthase is activated by multiple divalent metal cations, some of which have significant intracellular concentrations and some of which dissociate slowly. The nature of DAHP oxime's competition with the metal ion was investigated. Inhibition shifted from metal-competitive at physiological pH to metal-noncompetitive at pH > 8.7 in response to deprotonation of the Cys61 side chain. The modes of inhibition of DAHP synthase mutants and inhibitor fragments demonstrated that metal-competitive inhibition arose from interactions between Mn²⁺, DAHP oxime's O4 hydroxyl group, and the Cys61 and Asp326 side chains. The majority of potent DAHP synthase inhibitors in the literature possess a 4-hydroxyl group. Removing it could avoid metal-competitive inhibition and avoid them being outcompeted by metal ions in vivo.