Effect of subunit interactions on enzymatic activity of glutathione S-transferases: A radiation inactivatlon study

The glutathione S-transferases are a family of dimeric enzymes. Three isozymes from the alpha family, termed Y_aY_a, Y_aY_c, and Y_cY_c, and three from the mu family, termed Y_b1Y_b1, Y_b1Y_b2, and Y_b2Y_b2, were purified from rat liver. Binding studies were performed by equilibrium dialysis using a radiolabeled product, S-[¹⁴C](dinitrophenyl)glutathione. Each isozyme contained two independent binding sites which had equal affinity for the ligand. The presence of two independent active sites per enzyme dimer suggests that each subunit contains a complete active site. This conclusion was examined further using radiation inactivation which also allowed for assessment of the importance of subunit interactions in catalytic activity. The activity target size of Y_aY_a (47 kDa) was significantly larger than the protein monomer target size (31 kDa); similarly the activity target size of Y_aY_c was that of the dimer (54 kDa). In contrast, the activity target sizes of Y_b1Y_b1 and Y_b2Y_b2 were the same, being 35 and 29 kDa, respectively, and the protein monomer target size of Y_b1Y_b1 also was similar, being 32 kDa. These data indicate that interactions between subunits are critical for the maintenance of enzymatic activity of alpha class enzymes whereas each subunit of the two mu class proteins is capable of independent catalytic activity.