Kinetic evaluation of glucose 1-phosphate analogues with a thymidylyltransferase using a continuous coupled enzyme assay

Org Biomol Chem. 2015 Jan 21;13(3):866-75. doi: 10.1039/c4ob02057j.

Abstract

Cps2L, a thymidylytransferase, is the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis and an antibacterial target. We herein report the evaluation of six sugar phosphate analogues selected to further probe Cps2L substrate tolerance. A modified continuous spectrophotometric assay was employed for facile detection of pyrophosphate (PPi) released from nucleotidylyltransfase-catalysed condensation of sugar 1-phosphates and nucleoside triphosphates to produce sugar nucleotides. Additionally, experiments using waterLOGSY NMR spectroscopy were investigated as a complimentary method to evaluate binding affinity to Cps2L.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / chemical synthesis
  • Anti-Bacterial Agents / chemistry*
  • Bacterial Proteins / antagonists & inhibitors
  • Bacterial Proteins / chemistry*
  • Diphosphates / analysis
  • Enzyme Assays
  • Enzyme Inhibitors / chemical synthesis
  • Enzyme Inhibitors / chemistry*
  • Glucosephosphates / chemistry*
  • Kinetics
  • Nucleotidyltransferases / antagonists & inhibitors
  • Nucleotidyltransferases / chemistry*
  • Recombinant Proteins / chemistry
  • Spectrophotometry
  • Streptococcus pneumoniae / chemistry
  • Streptococcus pneumoniae / enzymology

Substances

  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Diphosphates
  • Enzyme Inhibitors
  • Glucosephosphates
  • Recombinant Proteins
  • diphosphoric acid
  • glucose-1-phosphate
  • Nucleotidyltransferases
  • glucose-1-phosphate thymidylyltransferase