Electrostatic interactions affecting the active site of class Sigma glutathione S-transferase

We have shown previously that the solvent-induced equilibrium unfolding mec hanism of class Sigma glutathione S-transferase (GST) is strongly affected by ionic strength [Stevens, Hornby, Armstrong and Dirr (1998) Biochemistry 37, 15534-15541]. The protein is dimeric and has a hydrophilic subunit inte rface. Here we show that ionic strength alone has significant effects on th e conformation of the protein, in particular at the active site. With the u se of NaCl at up to 2 M under equilibrium conditions, the protein lost 60 % of its catalytic activity and the single tryptophan residue per subunit be came partly exposed. The effect was independent of protein concentration, e liminating the dissociation of the dimer as a possibility for the conformat ional changes. This was confirmed by size-exclusion HPLC. There was no sign ificant change in the secondary structure of the protein according to far-U V CD data. Manual-mixing and stopped-flow kinetics experiments showed a slo w single-exponential salt-induced change in protein fluorescence. For equil ibrium and kinetics experiments, the addition of an active-site ligand (S-h exylglutathione) completely protected the protein from the ionic-strength-i nduced conformational changes. This suggests that the change occurs at or n ear the active site. Possible structural reasons for these novel effects ar e proposed, such as the flexibility of the alpha-helix 2 region as well as the hydrophilic subunit interface, highlighting the importance of electrost atic interactions in maintaining the structure of the active site of this G ST.