Phosphoenol acetylphosphonates: Substrate analogues as inhibitors of phosphoenolpyruvate enzymes

Abstract

Phosphoenol acetylphosphonate (1) and its bis methyl ester (2) are structurally and electrostatically defined analogues of phosphoenolpyruvate (PEP) in which the COO − moiety of PEP is replaced by a P(O)O −(OR) group. It is expected that such materials will bind to electrostatic receptors but the stereoelectronic properties of the phosphonate group will align the molecule at an angle that differs from that of the carboxylate substrate. ▪ The ability of these analogues to bind in place of PEP was evaluated with three enzymes that utilize PEP as a substrate in distinct reactions. Both 1 and 2 inhibit enolase and PEP carboxylase but they do not bind to pyruvate kinase. The inhibition of enolase at pH 6.8 is competitive with respect to 2-phosphoglycerate (the substrate in the reverse of the reaction that utilizes PEP) with K i = 2.2 ± 0.4 m m for 1 and 27 ± 4 m m for 2. The inhibition of enolase by 1 follows a titration curve consistent with the trianionic form of 1 being the active inhibitory form of the analogue. The specificity for the trianionic form shows that the compound is not an analogue of the aci-carboxylate reaction intermediate, which is a tetraanion. Neither 1 nor 2 are substrates for any of the enzymes. Inhibition of PEP carboxylase is pH dependent: at pH 7.8, 1 is a competitive inhibitor of the enzyme with respect to PEP ( K i = 2.2 ± 0.6 m m) while at pH 6.8 it is an activator; 2 has no effect on this enzyme. The diversity of the effects of the phosphonate analogues is consistent with the existence of distinct classes of binding sites for PEP. The variation in binding patterns suggests that the relationship of the points of contact of the substrate with the enzyme and with other substrates differs markedly in each of the enzymes studied.