Heterodimers of wild-type and subunit interface mutant enzymes of glutathione S-transferase A1-1: interactive or independent active sites?
Heterodimers of rat glutathione S-transferase A1-1 were formed using one wild-type subunit and one subunit with a mutation at the interface to evaluate whether the subunits are interactive or independent. Within the subunit interface, we are considering two regions of interactions: one region consists of a "hydrophobic ball and socket" with Phe 52 from one subunit as the ball and Phe 136 from the second subunit as one of the socket residues. The second region of interaction consists of Arg 69 and Glu 97 from both subunits. The heterodimers were formed after incubation in 1,6-hexanediol. Because one subunit in each pair had a His-tag, the heterodimers were purified using a nickel-nitrilotriacetic acid column. The specific activities of the heterodimer were compared with those of the two homodimers to determine whether the less active, mutant subunit communicates with the other subunit. Two of the heterodimers, wild type/R69E-His and wild type/E97Q-His, displayed specific activities much lower than that expected for independent active sites; in these cases, there are new close repulsive interactions and the low activity of one subunit is communicated to the neighboring subunit. In contrast, the other two heterodimers, wild type/R69Q-His and F136A/wild type-His, exhibited specific activities similar to those expected for independent active sites; in these heterodimers, the closest interaction is not repulsive or occurs over a much longer distance and the subunits act independently. We conclude that whether the subunits interact or are independent depends on the nature of the interactions at the subunit interface.
Vargo
Melissa A
MA
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Colman
Roberta F
RF
D000818:Animals; D001665:Binding Sites; D019281:Dimerization; D015870:Gene Expression; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007527:Isoenzymes; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D007527:Isoenzymes; D021122:Protein Subunits; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha