Catalytically active monomer of glutathione S-transferase pi and key residues involved in the electrostatic interaction between subunits.
Human glutathione transferase pi (GST pi) has been crystallized as a homodimer, with a subunit molecular mass of approximately 23 kDa; however, in solution the average molecular mass depends on protein concentration, approaching that of monomer at <0.03 mg/ml, concentrations typically used to measure catalytic activity of the enzyme. Electrostatic interaction at the subunit interface greatly influences the dimer-monomer equilibrium of the enzyme and is an important force for holding subunits together. Arg-70, Arg-74, Asp-90, Asp-94, and Thr-67 were selected as target sites for mutagenesis, because they are at the subunit interface. R70Q, R74Q, D90N, D94N, and T67A mutant enzymes were constructed, expressed in Escherichia coli, and purified. The construct of N-terminal His tag enzyme facilitates the purification of GST pi, resulting in a high yield of enzyme, but does not alter the kinetic parameters or secondary structure of the enzyme. Our results indicate that these mutant enzymes show no appreciable changes in K(m) for 1-chloro-2,4-dinitrobenzene and have similar CD spectra to that of wild-type enzyme. However, elimination of the charges of either Arg-70, Arg-74, Asp-90, or Asp-94 shifts the dimer-monomer equilibrium toward monomer. In addition, replacement of Asp-94 or Arg-70 causes a large increase in the K(m)(GSH), whereas substitution for Asp-90 or Arg-74 primarily results in a marked decrease in V(max). The GST pi retains substantial catalytic activity as a monomer probably because the glutathione and electrophilic substrate sites (such as for 1-chloro-2,4-dinitrobenzene) are predominantly located within each subunit.
Huang
Yu-chu
YC
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Misquitta
Stephanie
S
Blond
Sylvie Y
SY
Adams
Elizabeth
E
Colman
Roberta F
RF
D000595:Amino Acid Sequence; D002384:Catalysis; D002942:Circular Dichroism; D019281:Dimerization; D051549:Glutathione S-Transferase pi / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity
D051549:Glutathione S-Transferase pi
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