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

Abstract

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, Mn 2+ , 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 Mn 2+ , 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.

NeuNAc Oxime: A Slow-Binding and Effectively Irreversible Inhibitor of the Sialic Acid Synthase NeuB

“…In cases of partial inhibition, i.e., residual activity at high inhibition concentrations, Eadie−Hofstee plots converge toward the y-axis at high substrate concentrations but become nonlinear at low substrate concentrations. 9 For both PEP and ManNAc, the Eadie− Hofstee plots were linear at high substrate concentrations and the lines converged toward the y-axis, supporting competitive inhibition with respect to both substrates. This was supported by the crystal structure, which showed NeuNAc oxime bound in the catalytic site (see below), meaning that inhibition is necessarily competitive with respect to both substrates.…”

“…Eadie–Hofstee plots demonstrated that NeuNAc oxime’s mode of inhibition was competitive with respect to PEP and ManNAc (Figure ). In cases of partial inhibition, i.e., residual activity at high inhibition concentrations, Eadie–Hofstee plots converge toward the y -axis at high substrate concentrations but become nonlinear at low substrate concentrations . For both PEP and ManNAc, the Eadie–Hofstee plots were linear at high substrate concentrations and the lines converged toward the y -axis, supporting competitive inhibition with respect to both substrates.…”

“…In addition, competitive inhibition with respect to Mn 2+ was observed for DAHP synthase inhibition by DAHP oxime, even though the inhibitor does not occupy the same physical space as the metal ion. 7,9 This could be a common, though not universal, 8 feature of α-carboxyketose synthase inhibition.…”

“…The inset shows [S] vs v 0 /[E] 0 data. The curvature in the Eadie–Hofstee plots at low rates is due to the effects of partial inhibition; competitive inhibitors give lines that converge toward the y -axis at high rates. (c) Apparent K M,Mn values as a function of inhibitor concentration.…”

“…It catalyzes the metal ion-dependent condensation of N -acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP), passing through a tetrahedral intermediate (THI) to form NeuNAc (Figure ). It is the archetypal member of the NeuB superfamily of α-carboxyketose synthases, which includes 2-keto-3-deoxy- d - arabino -heptulosonate-7-phosphate (DAHP) and 2-keto-3-deoxy- d - manno -octulosonate-8-phosphate (KDO8P) synthases, − which are also antimicrobial targets. DAHP synthase ,, and KDO8P synthase , follow sequential ordered ter ter kinetic mechanisms, which is likely universal throughout the NeuB superfamily.…”