Crystal structure of gamma-glutamylcysteine synthetase: insights into the mechanism of catalysis by a key enzyme for glutathione homeostasis

Proc Natl Acad Sci U S A. 2004 Oct 19;101(42):15052-7. doi: 10.1073/pnas.0403277101. Epub 2004 Oct 11.

Abstract

Gamma-glutamylcysteine synthetase (gammaGCS), a rate-limiting enzyme in glutathione biosynthesis, plays a central role in glutathione homeostasis and is a target for development of potential therapeutic agents against parasites and cancer. We have determined the crystal structures of Escherichia coli gammaGCS unliganded and complexed with a sulfoximine-based transition-state analog inhibitor at resolutions of 2.5 and 2.1 A, respectively. In the crystal structure of the complex, the bound inhibitor is phosphorylated at the sulfoximido nitrogen and is coordinated to three Mg2+ ions. The cysteine-binding site was identified; it is formed inductively at the transition state. In the unliganded structure, an open space exists around the representative cysteine-binding site and is probably responsible for the competitive binding of glutathione. Upon inhibitor binding, the side chains of Tyr-241 and Tyr-300 turn, forming a hydrogen-bonding triad with the carboxyl group of the inhibitor's cysteine moiety, allowing this moiety to fit tightly into the cysteine-binding site with concomitant accommodation of its side chain into a shallow pocket. This movement is caused by a conformational change of a switch loop (residues 240-249). Based on this crystal structure, the cysteine-binding sites of mammalian and parasitic gammaGCSs were predicted by multiple sequence alignment, although no significant sequence identity exists between the E. coli gammaGCS and its eukaryotic homologues. The identification of this cysteine-binding site provides important information for the rational design of novel gammaGCS inhibitors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Catalysis
  • Crystallography, X-Ray
  • Cysteine / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Glutamate-Cysteine Ligase / chemistry*
  • Glutamate-Cysteine Ligase / genetics
  • Glutamate-Cysteine Ligase / metabolism
  • Glutamic Acid / metabolism
  • Glutathione / metabolism
  • Homeostasis
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Static Electricity

Substances

  • Recombinant Proteins
  • Glutamic Acid
  • Glutamate-Cysteine Ligase
  • Glutathione
  • Cysteine

Associated data

  • PDB/1V4G
  • PDB/1VAG