The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases

Claire S Allardyce, Paul D McDonagh, Lu-Yun Lian, C R Wolf, G C Roberts

Research output: Contribution to journalArticlepeer-review

53 Citations (Scopus)

Abstract

Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the 'essential' Tyr(9) by phenylalanine leads to a marked decrease in the k(cat) for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on k(cat) for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in k(cat) for CDNB, but an increase in k(cat) for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity ('turnover number') for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change.

Original languageEnglish
Pages (from-to)525-31
Number of pages7
JournalThe Biochemical journal
Volume343
Issue number3
DOIs
Publication statusPublished - 1 Nov 1999

Keywords

  • Amino Acid Substitution
  • Binding Sites
  • Catalysis
  • Dinitrochlorobenzene/pharmacokinetics
  • Ethacrynic Acid/pharmacokinetics
  • Glutathione Transferase/chemistry
  • Isoenzymes/chemistry
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Phenylalanine
  • Protein Structure, Secondary
  • Recombinant Proteins/chemistry
  • Substrate Specificity
  • Tyrosine

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