Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1

Abstract

The "mu loop," an 11-residue loop spanning amino acid residues 33-43, is a characteristic structural feature of the mu class of glutathione transferases. To assess the contribution of the mu loop to the structure and function of rat GST M1-1, amino acid residues 35-44 (35GDAPDYDRSQ44) were excised by deletion mutagenesis, resulting in the "Deletion Enzyme." Kinetic studies reveal that the Km values of the Deletion Enzyme are markedly increased compared with those of the wild-type enzyme: 32-fold for 1-chloro-2,4-dinitrobenzene, 99-fold for glutathione, and 880-fold for monobromobimane, while the Vmax value for each substrate is increased only modestly. Results from experiments probing the structure of the Deletion Enzyme, in comparison with that of the wild-type enzyme, suggest that the secondary and quaternary structures have not been appreciably perturbed. Thermostability studies indicate that the Deletion Enzyme is as stable as the wild-type enzyme at 4 degrees C and 10 degrees C, but it rapidly loses activity at 25 degrees C, unlike the wild-type enzyme. In the temperature range of 4 degrees C through 25 degrees C, the loss of activity of the Deletion Enzyme is not the result of a change in its structure, as determined by circular dichroism spectroscopy and sedimentation equilibrium centrifugation. Collectively, these results indicate that the mu loop is not essential for GST M1-1 to maintain its structure nor is it required for the enzyme to retain some catalytic activity. However, it is an important determinant of the enzyme's affinity for its substrates.

Figure 1.

(A) Structure of the dimeric form of GST M1-1 (PDB code 6GST; Xiao et al. 1996). (Cyan) Subunit A; (white) subunit B. The mu loop of each subunit, amino acid residues 33–43 as well as the amino acid residue 44, is highlighted in red. (B) Structure of one subunit of the dimer of GST M1-1 (PDB code 6GST). The mu loop, amino acid residues 33–43, is highlighted in red. GSH is shown as it was crystallized with the wild-type enzyme; GSH is colored in green. Yellow arrows show the approximate location of the alpha carbon of amino acid residues 35 and 44. The monomer has been rotated 90° around the Y-axis to provide a better view of the mu loop.

Figure 2.

The steady-state fluorescence spectra of the wild-type enzyme and the Deletion Enzyme (2.2 × 10⁻⁵ M enzyme subunits) in 0.1 M potassium phosphate buffer (pH 6.5) containing 1 mM EDTA at 4°C were measured on a Perkin Elmer MPF-3 fluorescence spectrophotometer. Emission spectrum (excitation at 280 nm, bandwidth of 10 nm) of GST M1-1 (•), the Deletion Enzyme (▾), and free tryptophan (▪).

Figure 3.

Thermostability study of the wild-type enzyme and the Deletion Enzyme at 4°C, 10°C, and 25°C. In each panel, wild-type enzyme is represented by the black triangles (▴) connected by a dashed line and the Deletion Enzyme is represented by the black circles (•) connected by a solid line. The rates of inactivation of the wild-type enzyme and the Deletion Enzyme were monitored at 4°C (A) 10°C (B) and 25°C (C). The activity of the enzymes is expressed as E_t/E_o (observed activity/initial activity).

Figure 4.

pH dependence of k_cat/K_m^CDNB for the wild-type enzyme (A) and the Deletion Enzyme (B). These values were determined by fitting the data to the equation pK_a = (k_cat/K_m^CDNB)/(1 + 10^(pKa−pH)), where k_cat is the turnover number of the enzyme for the substrates GSH and CDNB, the K_m is that of CDNB, and the pH is that at which the catalytic measurements were recorded.

Figure 5.

(A) Overlay of GST M1-1 (6GST, white) and the homology model of the Deletion Enzyme (blue) showing the difference in solvent exposure of Trp45 (GST M1-1, red) and Trp35 (Deletion Enzyme, yellow). The mu loop of the wild-type enzyme is shown in red and the remaining mu loop region of the Deletion Enzyme is shown in yellow. Please note that amino acid residue 45 of wild-type enzyme is equivalent to amino acid residue 35 of the Deletion Enzyme. (In the wild-type enzyme residues 45 to 217 are equivalent to those numbered 10 less in the Deletion Enzyme. Specifically, Trp45 of the wild-type enzyme is equivalent to Trp35 of the Deletion Enzyme. The other tryptophan residues present in each enzyme are also displayed. GSH is shown as bound in wild-type enzyme and is colored green. (B) Enlargement of the active site region of the wild-type enzyme and the Deletion Enzyme as shown in A. The coloring remains the same with the exception that the GSH molecule is colored by atom (oxygen, red; nitrogen, blue; sulfur, yellow; carbon, white).

Figure 6.

Enlargement of the active site of the wild-type enzyme and the Deletion Enzyme showing the proximity of Leu36, Lys39, and Leu49 of the Deletion Enzyme and Leu46, Lys 49, and Leu59 of the wild-type enzyme to the glycyl moiety of GSH. The wild-type enzyme's amino acid residues and its mu loop region are shown in red, the Deletion Enzyme's amino acid residues and its remaining mu loop region are shown in yellow, and GSH is colored by atom (oxygen, red; nitrogen, blue; sulfur, yellow; carbon, white). Please note that amino acid residue 49 of wild-type enzyme is equivalent to amino acid residue 39 of the Deletion enzyme. (In the wild-type enzyme residues 45 to 217 are equivalent to those numbered 10 less in the Deletion Enzyme. Specifically, Trp45 of the wild-type enzyme is equivalent to Trp35 of the Deletion Enzyme.)