Delineation of xenobiotic substrate sites in rat glutathione S-transferase M1-1

Abstract

Glutathione S-transferases catalyze the conjugation of glutathione with endogenous and exogenous xenobiotics. Hu and Colman (1995) proposed that there are two distinct substrate sites in rat GST M1-1, a 1-chloro-2,4-dintrobenzene (CDNB) substrate site located in the vicinity of tyrosine-115, and a monobromobimane (mBBr) substrate site. To determine whether the mBBr substrate site is distinguishable from the CDNB substrate site, we tested S-(hydroxyethyl)bimane, a nonreactive derivative of mBBr, for its ability to compete kinetically with the substrates. We find that S-(hydroxyethyl)bimane is a competitive inhibitor (K(I) = 0.36 microM) when mBBr is used as substrate, but not when CDNB is used as substrate, demonstrating that these two sites are distinct. Using site-directed mutagenesis, we have localized the mBBr substrate site to an area midway through alpha-helix 4 (residues 90-114) and have identified residues that are important in the enzymatic reaction. Substitution of alanine at positions along alpha-helix 4 reveals that mutations at positions 103, 104, and 109 exhibit a greater perturbation of the enzymatic reaction with mBBr than with CDNB as substrate. Various other substitutions at positions 103 and 104 reveal that a hydrophobic residue is necessary at each of these positions to maintain optimal affinity of the enzyme for mBBr and preserve the secondary structure of the enzyme. Substitutions at position 109 indicate that this residue is important in the enzyme's affinity for mBBr but has a minimal effect on Vmax. These results demonstrate that the promiscuity of rat GST M1-1 is in part due to at least two distinct substrate sites.

Figure 1.

A comparison of the alanine mutant enzymes’ kinetic parameters. The CDNB kinetic parameters are represented by black vertical bars, and the mBBr kinetic parameters are represented by gray vertical bars. (A) Ratio of mutant GST M1-1 V_max to wild-type GST M1-1 V_max. (B) Ratio of mutant GST M1-1 K_m to wild-type GST M1-1 K_m.

Figure 2.

A comparison of the M104 mutant enzymes’ kinetic parameters. The CDNB kinetic parameters are represented by black vertical bars, and the mBBr kinetic parameters are represented by gray vertical bars. (A) Ratio of mutant GST M1-1 V_max to wild-type GST M1-1 V_max. (B) Ratio of mutant GST M1-1 K_m to wild-type GST M1-1 K_m. Please note that the magnitude of the Y-axis is different from that of Figure 1 ▶.

Figure 3.

A comparison of the Q109 mutant enzymes’ kinetic parameters. The CDNB kinetic parameters are represented by black vertical bars, and the mBBr kinetic parameters are represented by gray vertical bars. (A) Ratio of mutant GST M1-1 V_max to wild-type GST M1-1 V_max. (B) Ratio of mutant GST M1-1 K_m to wild-type GST M1-1 K_m. Please note that the magnitude of the Y-axis is different from that of Figures 1 ▶ and 2 ▶.

Figure 4.

A comparison of the V103 mutant enzymes’ kinetic parameters. The CDNB kinetic parameters are represented by black vertical bars, and the mBBr kinetic parameters are represented by gray vertical bars. (A) Ratio of mutant GST M1-1 V_max to wild-type GST M1-1 V_max. (B) Ratio of mutant GST M1-1 K_m to wild-type GST M1-1 K_m. Please note that the magnitude of the Y-axis is different from those of Figures 1 ▶–3 ▶ ▶.

Figure 5.

A comparison of the C114A and Y115F mutant enzymes’ kinetic parameters. The CDNB kinetic parameters are represented by black vertical bars, and the mBBr kinetic parameters are represented by gray vertical bars. (A) Ratio of mutant GST M1-1 V_max to wild-type GST M1-1 V_max. (B) Ratio of mutant GST M1-1 K_m to wild-type GST M1-1 K_m. Please note that the magnitude of the Y-axis is different from those of Figures 1 ▶–4 ▶ ▶ ▶.

Figure 6.

(A) Model of GST M1-1 with the monobromobimane molecule docked in the experimentally defined monobromobimane substrate site. The mBBr molecule is colored by atom (red for oxygen, blue for nitrogen). Amino acid residues (A) V103, (A) M104, (B) Q109, and (A) Y115 (seen on edge) as well as the S-methylglutathione molecule (yellow) are colored in a solid color. The amino acid residues shown are: Val 103 (green), Met 104 (red), Gln 109 (purple), and Tyr 115 (gray). Subunit A is shown as a cyan ribbon with α-helix 4 accentuated in royal blue. Subunit B is shown as a pink ribbon with α-helix 4 accentuated in gray. (B) In silico model of the M104A mutant enzyme. (C) In silico model of the M104W mutant enzyme.

Figure 6.

(A) Model of GST M1-1 with the monobromobimane molecule docked in the experimentally defined monobromobimane substrate site. The mBBr molecule is colored by atom (red for oxygen, blue for nitrogen). Amino acid residues (A) V103, (A) M104, (B) Q109, and (A) Y115 (seen on edge) as well as the S-methylglutathione molecule (yellow) are colored in a solid color. The amino acid residues shown are: Val 103 (green), Met 104 (red), Gln 109 (purple), and Tyr 115 (gray). Subunit A is shown as a cyan ribbon with α-helix 4 accentuated in royal blue. Subunit B is shown as a pink ribbon with α-helix 4 accentuated in gray. (B) In silico model of the M104A mutant enzyme. (C) In silico model of the M104W mutant enzyme.