Minor modifications of the C-terminal helix reschedule the favored chemical reactions catalyzed by theta class glutathione transferase T1-1

Abstract

Adaptive responses to novel toxic challenges provide selective advantages to organisms in evolution. Glutathione transferases (GSTs) play a pivotal role in the cellular defense because they are main contributors to the inactivation of genotoxic compounds of exogenous as well as of endogenous origins. GSTs are promiscuous enzymes catalyzing a variety of chemical reactions with numerous alternative substrates. Despite broad substrate acceptance, individual GSTs display pronounced selectivities such that only a limited number of substrates are transformed with high catalytic efficiency. The present study shows that minor structural changes in the C-terminal helix of mouse GST T1-1 induce major changes in the substrate-activity profile of the enzyme to favor novel chemical reactions and to suppress other reactions catalyzed by the parental enzyme.

FIGURE 1.

Comparison of wild type mouse GST T1-1 (mGST T1-1) and human GST T1-1 (hGST T1-1) sequences. The different α-helix regions indicated are based on the crystal structure of hGST T1-1 (Protein Data Bank code 2c3n) (24). H-site residues are marked with gray boxes. The G-site residues (not indicated) are conserved between human and mouse GST T1-1.

FIGURE 2.

Structures of alternative electrophilic substrates used in activity measurements. Arrows indicate the site of attack on the substrates: EPNP, NPB, phenethyl-ITC, allyl-ITC, propyl-ITC, benzyl-ITC, H₂O₂, tert-BuOOH, and CuOOH. See supplemental Table S1 for assay conditions.

FIGURE 3.

Pyramid plot of catalytic activity profiles of wild type mouse GST T1-1 and variants M232A, M232A/R234W, R234W, and ΔR234W with 18 alterative electrophilic substrates. The substrates used were EPNP, NPB, phenethyl-ITC, allyl-ITC, propyl-ITC, benzyl-ITC, H₂O₂, tert-BuOOH, CuOOH, and different (mono- and di-) iodoalkanes. The specific activity values are given in supplemental Table S2.

FIGURE 4.

Double mutant cycle of two residues in the C-terminal helix influencing the activity profile in GST T1-1. The upper part shows a scheme of the two substitutions in GST T1-1 in the Met²³² and Arg²³⁴ positions. The lower part shows the ratios of mutant activities to the corresponding wild type activities for alternative substrates.

FIGURE 5.

Comparison of the C-terminal helix in wild type mouse GST T1-1 and the M232A and ΔR234W variants. Modeled structures of mouse GST T1-1 variants (red) are superpositioned with the W234R mutant of human GST T1-1 (blue) in complex with S-hexylglutathione (Protein Data Bank code 2c3q). A, wild type mouse GST T1-1. B, mutant M232A. C, mutant ΔR234W. The amino acids Met²³², Pro²³³, Arg²³⁴, Val²³⁵, and Leu²³⁶ are shown in ball and stick representation. In mutant ΔR234W, the numbering of Pro and Trp (red) has changed because of the deletion of Met²³². The figure was produced using the University of California, San Francisco, Chimera program.

FIGURE 6.

Rescheduling of favored chemical reactions by point mutations in GST T1-1. Pie charts show the relative activities of wild type GST T1-1 and the four variants R234W, M232A, ΔR234W, and M232A/R232W with the alternative substrates EPNP, iodomethane, CuOOH, and allyl-ITC, representing four diverse chemical reactions.