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Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases
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Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

Published on: October 10, 2020

Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive

H Cheng1, T Tchaikovskaya, Y S Tu

  • 1Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.

The Biochemical Journal
|May 23, 2001
PubMed

Insights

Researchers expressed recombinant rat glutathione S-transferase M4-4 (rGSTM4-4) protein. This enzyme showed lower catalytic efficiency but unique redox activity at Cys-115, suggesting specialized roles in oxidative stress.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • The rat glutathione S-transferase (GST) M4 (rGSTM4) gene is known, but its protein product has not been purified from tissue.
  • Glutathione S-transferases are crucial enzymes involved in detoxification and cellular defense against oxidative stress.

Purpose of the Study:

  • To express and characterize recombinant rat glutathione S-transferase M4-4 (rGSTM4-4) protein.
  • To investigate the catalytic properties, structural features, and potential physiological functions of rGSTM4-4.

Main Methods:

  • Chemical synthesis of the rGSTM4 gene and expression in Escherichia coli.
  • Enzyme kinetics assays using 1-chloro-2,4-dinitrobenzene (CDNB) conjugation.
  • Site-directed mutagenesis (C115Y) and molecular modeling.
  • Antibody-based detection of rGSTM4-4 in rat and mouse liver.

Main Results:

  • Recombinant rGSTM4-4 exhibited significantly lower catalytic efficiency (k(cat)/K(m)) for CDNB conjugation compared to rGSTM1-1, with a higher pH optimum.
  • A unique Cys-115 residue in rGSTM4-4 is redox-reactive, forming an active S-glutathiolated enzyme.
  • Mutation of Cys-115 to Tyr (C115Y) increased catalytic efficiency 6-10 fold.
  • Trp-45 is essential for rGSTM4-4 activity and glutathione binding.
  • An orthologous mouse GSTM4-4 was detected and found to be inducible in mouse liver.

Conclusions:

  • Rodent Mu-class GSTs, like rGSTM4-4, possess unique structural features, including a redox-active Cys-115.
  • These structural characteristics suggest specialized physiological roles, potentially in managing oxidative stress.
  • The inducibility of mouse GSTM4-4 indicates its involvement in adaptive cellular responses.

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