On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer

Abstract

Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3(-) complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3 on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.

Sodium Fluoride Exposure Leads to ATP Depletion and Altered RNA Decay in Escherichia coli under Anaerobic Conditions

“…It has long been known that fluoride ions can interrupt glycolysis by inhibiting the activities of the glycolytic enzyme, enolase ( 6 ) and the gluconeogenetic enzyme, phosphoenolpyruvate synthetase ( 7 ). Experiments on eukaryotic cells have shown that inhibition of these enzymes reduces ATP levels ( 8 ).…”

“…This metabolic pathway is particularly essential under the anaerobic conditions encountered by E. coli and other pathogenic bacteria in their animal hosts. It has long been known that fluoride ions inhibit the activities both the glycolytic enzyme enolase ( 6 ) and gluconeogenetic enzyme phosphoenolpyruvate synthetase ( 7 ). Enolase is a key glycolytic enzyme involved in converting 2-glycerol phosphate to phosphoenolpyruvate ( 5 ).…”

Biotechnology Applications of Cell-Free Expression Systems

“…One of these experiments demonstrated that adding cofactors into the reaction mix that inhibit or activate enzymes and pathways that directly interact with the protein synthesis reaction can improve yields and synthesis duration. The cofactors included: increasing the amount of ATP produced in reaction by adding additional factors to react with pyruvate; increasing the concentration of amino acids in the reaction mix to prevent resource exhaustion; and the inhibition of the futile cycle of PEP synthetase via oxalate addition which prevented conversion of phosphoenolpyruvate product back into pyruvate precursor [35]. Another experiment showed an enzyme-based cell-free platform that exhibited CFPS reactions in two separate formats: using purified components rather than a cell lysate and coupled transcription/translation [15].…”

A single phosphorylation mechanism in early metabolism – the case of phosphoenolpyruvate

“…In the enzymatic mechanism, the enolate of pyruvate is generated within the active site of the enzyme (stabilized by Mg 2+ ), which subsequently undergoes phosphoryl transfer from a phosphohistidine intermediate to yield PEP. [32][33][34] In the following steps of anabolic pathways, PEP provides all carbon and phosphoryl groups for the sugar-phosphate intermediates of gluconeogenesis and the pentose phosphate pathway, including ribose-5phosphate, the building block for ribonucleotide biosynthesis. Thus, enzymatic phosphorylation serves not only a bioenergetic role but also a fundamental structural role in biochemistry through PEP.…”