Hidden allostery in human glutathione transferase p1-1 unveiled by unnatural amino acid substitutions and inhibition studies

J Mol Biol. 2013 May 13;425(9):1509-14. doi: 10.1016/j.jmb.2013.01.038. Epub 2013 Feb 8.

Abstract

Conventional steady-state kinetic studies of the dimeric human glutathione transferase (GST) P1-1 do not reveal obvious deviations from Michaelis-Menten behavior. By contrast, engineering of the key residue Y50 of the lock-and-key motif in the subunit interface reveals allosteric properties of the enzyme. The low-activity mutant Y50C, characterized by 150-fold decreased kcat and 300-fold increased KM(GSH) values, displays an apparent Hill coefficient of 0.82±0.22. Chemical alkylation of the sulfhydryl group of Y50C by unnatural n-butyl or n-pentyl substitutions enhances the catalytic efficiency kcat/KM(GSH) to near the wild-type value but still yields Hill coefficients of 0.61±0.08 and 0.86±0.1, respectively. Thus, allosteric kinetic behavior is not dependent on low activity of the enzyme. On the other hand, S-cyclobutylmethyl-substituted Y50C, which also displays high catalytic efficiency, has a Hill coefficient of 0.99±0.11, showing that subtle differences in structure at the subunit interface influence the complex kinetic behavior. Furthermore, inhibition studies of native GST P1-1 using ethacrynic acid demonstrate that a ligand bound noncovalently to the wild-type enzyme also can elicit allosteric kinetic behavior. Thus, we conclude that the GST P1-1 structure has intrinsic allostery that becomes overt under some, but not all, ambient conditions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Regulation / genetics
  • Allosteric Site / genetics
  • Amino Acid Substitution / genetics
  • Glutathione S-Transferase pi / antagonists & inhibitors
  • Glutathione S-Transferase pi / chemistry*
  • Glutathione S-Transferase pi / metabolism*
  • Humans
  • Models, Molecular
  • Protein Subunits / antagonists & inhibitors
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism

Substances

  • Protein Subunits
  • Glutathione S-Transferase pi