Linear Free Energy Relationship Analysis of Transition State Mimicry by 3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) Oxime, a DAHP Synthase Inhibitor and Phosphate Mimic

Abstract

3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime replaces the ketone, is a potent inhibitor, with K = 1.5 μM. Linear free energy relationship (LFER) analysis of DAHP oxime inhibition using DAHP synthase mutants revealed an excellent correlation between transition state stabilization and inhibition. The equations of LFER analysis were rederived to formalize the possibility of proportional, rather than equal, changes in the free energies of transition state stabilization and inhibitor binding, in accord with the fact that the majority of LFER analyses in the literature demonstrate nonunity slopes. A slope of unity, m = 1, indicates that catalysis and inhibitor binding are equally sensitive to perturbations such as mutations or modified inhibitor/substrate structures. Slopes <1 or >1 indicate that inhibitor binding is less sensitive or more sensitive, respectively, to perturbations than is catalysis. LFER analysis using the tetramolecular specificity constant, that is, plotting log(KKK/k) versus log(K), revealed a slope, m, of 0.34, with r = 0.93. This provides evidence that DAHP oxime is mimicking the first irreversible transition state of the DAHP synthase reaction, presumably phosphate departure from the tetrahedral intermediate. This is evidence that the oxime group can act as a functional, as well as structural, mimic of phosphate groups.

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

“…The oxime functional group appears to be a general inhibitory motif for α-carboxyketose synthases, as NeuB is now the third member to be inhibited by oximes. − , Because it mimics phosphate groups both structurally and functionally, the oxime group can be considered a small, neutral phosphate bioisostere in this context, even though it is not an isostere in the chemical sense . In the same way, carboxylates are considered phosphate bioisosteres even though they are not chemical isosteres.…”

“…MnCl 2 concentrations of 0–15 mM were tested but caused substrate inhibition above 2 mM and were not routinely used in fitting the kinetic parameters. Mn 2+ is an essential activator but was treated as a substrate in initial velocity calculations, as described previously. ,,, The kinetic parameters were determined by fitting all 67 initial velocity points simultaneously to the sequential ordered ter ter kinetic mechanism, as described previously (eq ), , so the reported standard errors include both intra- and interday variability. …”

“…17−21 The oxime functional group of DAHP oxime, plus two crystallographic waters, structurally mimics the THI's phosphate group and functionally mimics the transition state for THI breakdown. 16 We describe here the inhibition of N. meningitidis NeuB by NeuNAc oxime. It is a slow-onset noncovalent inhibitor that, once bound, has an unmeasurably long residence time.…”

“…α-Carboxyketose synthases have been the targets of inhibition since the 1970s; however, highly potent inhibitors remain rare, and the only potent, mechanism-based inhibitor of NeuB characterized to date is a slow-binding THI mimic ( 1 ) with a K i * of 3 μM (Figure , bottom) . We recently demonstrated that the DAHP oxime is a transition state mimic and potent inhibitor of DAHP synthase, with a K i of 1.5 μM, and a residence time in the active site, t R , of 83 min, , while KDO8P oxime inhibits KDO8P synthase with a K i * down to 0.57 μM and residence times of up to 69 h . Long residence times are an increasingly important inhibitor design criterion due to the correlation between slow dissociation and drug efficacy in vivo. − The oxime functional group of DAHP oxime, plus two crystallographic waters, structurally mimics the THI’s phosphate group and functionally mimics the transition state for THI breakdown …”

FDA-Approved Oximes and Their Significance in Medicinal Chemistry

“…Oximes also showed comparable results to ketones, in the studies of LTB4 inhibition. However, most importantly, oximes and oxime ethers can be used as analogs for phosphate groups and for peptide bonds [31][32][33]. Potentially, such bioisosterism can lead to the discoveries of new therapeutic agents for the treatment of nucleotide and protein-related illnesses.…”