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 c loned and expressed in Escherichia coli, and its catalytic mechanism was in vestigated by site-directed mutagenesis and dynamic studies. The results sh owed that the enzyme promotes proton dissociation from the GSH thiol and cr eates 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 modulati on between the GSH binding site (G-site) and the electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent, an d thermodynamic data suggest that product release is rate-limiting. Five re sidues of GST I (Ser(11), His(10), Lys(11), Gln(53) and Ser(67)), which are located in the G-site, were individually replaced with alanine and their s tructural and functional roles in the 1-chloro-2,4-dinitrobenzene (CDNB) co njugation reaction were investigated. On the basis of steady-state kinetics , difference spectroscopy and limited proteolysis studies it is concluded t hat these residues: (1) contribute to the affinity of the G-site for GSH, a s they are involved in side-chain interaction with GSH; (2) influence GSH t hiol ionization, and thus its reactivity; (3) participate in k(eat) regulat ion by affecting the rate-limiting step of the reaction, and (4) in the cas es of His(40), Lys(41) and Gln(53) play an important role in the structural integrity of, and probably in the flexibility of, the highly mobile short 3,,helical segment of alpha -helix 2 (residues 35-46), as shown by limited proteolysis experiments. These structural perturbations are probably transm itted to the H-site through changes in Phe(35) conformation. This accounts for the modulation of Km(CDXB) by His(40), Lys(41) and Gln(53), and also fo r the intrasubunit communication between the G- and If-sites. Computer simu lations using CONCOORD were applied to maize GST I monomer and dimer struct ures, each with bound lactoylglutathione, and the results were analysed by the essential dynamics technique. Differences in dynamics were found betwee n the monomer and the dimer simulations showing the importance of using the whole structure in dynamic analysis. The results obtained confirm that the short 3(10)-helical segment of alpha -helix 2 (residues 35-46) undergoes t he most significant structural rearrangements. These rearrangements are dis cussed in terms of enzyme catalytic mechanism.