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

Biochem Cell Biol. 2015 Jun;93(3):236-40. doi: 10.1139/bcb-2014-0153. Epub 2015 Jan 26.

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.

Keywords: MgF3−/AlF4−; complexes de fluorures métalliques; enzyme inhibition; fluoride; fluorure; inhibition enzymatique; metal fluoride complexes; phosphoenolpyruvate synthase; phosphoénolpyruvate synthase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aluminum Chloride
  • Aluminum Compounds / pharmacology
  • Chlorides / pharmacology
  • Enzyme Inhibitors / pharmacology*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / antagonists & inhibitors
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Fluorides / pharmacology*
  • Kinetics
  • Magnesium Compounds / pharmacology*
  • Phosphotransferases (Paired Acceptors) / antagonists & inhibitors*
  • Phosphotransferases (Paired Acceptors) / genetics
  • Phosphotransferases (Paired Acceptors) / metabolism*
  • Pyruvate Synthase / antagonists & inhibitors
  • Pyruvate Synthase / genetics
  • Pyruvate Synthase / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sodium Fluoride / pharmacology*

Substances

  • Aluminum Compounds
  • Chlorides
  • Enzyme Inhibitors
  • Escherichia coli Proteins
  • Magnesium Compounds
  • Recombinant Proteins
  • Aluminum Chloride
  • magnesium fluoride
  • Sodium Fluoride
  • Pyruvate Synthase
  • ppsA protein, E coli
  • Phosphotransferases (Paired Acceptors)
  • pyruvate, water dikinase
  • Fluorides
  • aluminum fluoride