Catalytically active monomer of glutathione S-transferase pi and key residues involved in the electrostatic interaction between subunits

Abstract

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.

FIGURE 1.

Dimeric structure (side view) of human GST π (PDB 9GSS) crystallized with S-hexylglutathione, which is colored green. This structure shows the electrostatic region between two subunits. Each individual subunit is colored-coded: the backbone of subunit A is shown in cyan, while that of subunit B is red. The residues selected for mutagenesis at the subunit interface are Arg-70, Arg-74, Asp-90, Asp-94, and Thr-67. The side chains of the A subunit amino acid residues are in yellow, whereas those of the B subunit are purple.

FIGURE 2.

An enlargement of the wild-type human GST π interface showing interacting residues between the two subunits. Subunit A is displayed in cyan and subunit B is in red. A, the distance shown is between Arg-74 (A) (yellow) and Asp-90 (B) (purple) (3.0 Å). B, an enlargement of wild-type GST π interface showing Thr-67, Arg-70, and Asp-94 from both subunits. Subunit A is displayed in cyan and subunit B is in red. The distances shown are between the closest oxygen of Asp-94 (A subunit) and the guanido group of Arg-70 (B subunit) (4.56 Å), and between Thr-67A (A subunit) and Asp-94 (B subunit) (2.91 Å).

FIGURE 3.

The ClustalW sequence alignment of three mammalian π class GST isozymes. The mGSTPi, pGSTPi, and hGSTPi sequences are representative and are for mouse, porcine, and human species, respectively. The amino acids selected for mutagenesis in this study are designated by bold letters.

FIGURE 4.

CD spectra were determined with the protein (0. 3 mg/ml) in 0.1 m potassium phosphate (pH 6.5) containing 1 mm EDTA. CD spectra for non-His tag wild-type and mutant enzymes: WT (▴), T67A (○), R70Q (▵), R74Q (•), D90N (⋄), D94N (□).

FIGURE 5.

AFM images of human wild-type GST π (from a solution of 0. 25 mg/ml). Image size 25 nm × 4.5 μm. Arrow 1: monomer; arrow 2: dimer. Inset: the calculated volume of proteins imaged by AFM plotted against the molecular mass. The proteins used are xylanase from T. longibraciatum (21.6 kDa), polygalacturonase from A. nigar (24.0 kDa), xylanase from C. japonicus (39.2 kDa), and bovine serum albumin (67.0 kDa).