Functional and structural roles of the glutathione-binding residues in maize (Zea mays) glutathione S-transferase I

The isoenzyme glutathione S-transferase (GST) I from maize (Zea mays) was cloned and expressed in Escherichia coli, and its catalytic mechanism was investigated by site-directed mutagenesis and dynamic studies. The results showed that the enzyme promotes proton dissociation from the GSH thiol and creates a thiolate anion with high nucleophilic reactivity by lowering the pK_a of the thiol from 8.7 to 6.2. Steady-state kinetics fit well to a rapid equilibrium, random sequential Bi Bi mechanism, with intrasubunit modulation between the GSH binding site (G-site) and the electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent, and thermodynamic data suggest that product release is rate-limiting. Five residues of GST I (Ser¹¹, His⁴⁰, Lys⁴¹, Gln⁵³ and Ser⁶⁷), which are located in the G-site, were individually replaced with alanine and their structural and functional roles in the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction were investigated. On the basis of steady-state kinetics, difference spectroscopy and limited proteolysis studies it is concluded that these residues: (1) contribute to the affinity of the G-site for GSH, as they are involved in side-chain interaction with GSH; (2) influence GSH thiol ionization, and thus its reactivity; (3) participate in k_cat regulation by affecting the rate-limiting step of the reaction; and (4) in the cases of His⁴⁰, Lys⁴¹ and Gln⁵³ play an important role in the structural integrity of, and probably in the flexibility of, the highly mobile short 3₁₀-helical segment of α-helix 2 (residues 35–46), as shown by limited proteolysis experiments. These structural perturbations are probably transmitted to the H-site through changes in Phe³⁵ conformation. This accounts for the modulation of K^cdnb_m by His⁴⁰, Lys⁴¹ and Gln⁵³, and also for the intrasubunit communication between the G- and H-sites. Computer simulations using CONCOORD were applied to maize GST I monomer and dimer structures, each with bound lactoylglutathione, and the results were analysed by the essential dynamics technique. Differences in dynamics were found between the monomer and the dimer simulations showing the importance of using the whole structure in dynamic analysis. The results obtained confirm that the short 3₁₀-helical segment of α-helix 2 (residues 35–46) undergoes the most significant structural rearrangements. These rearrangements are discussed in terms of enzyme catalytic mechanism.