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

Abstract

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.