Communication between the Two Active Sites of Glutathione S-Transferase A1-1, Probed Using Wild-Type−Mutant Heterodimers

• Published in: Biochemistry (Easton) Vol. 44; no. 24; pp. 8608 - 8619
• Main Authors: Misquitta, Stephanie A, Colman, Roberta F
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.06.2005
• Subjects: Amino Acid Substitution; Animals; Binding Sites; Circular Dichroism; Dimerization; Glutathione Transferase - chemistry; Glutathione Transferase - metabolism; Humans; Isoenzymes - chemistry; Isoenzymes - metabolism; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Protein Conformation; Protein Structure, Tertiary; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi050449a

To study the communication between the two active sites of dimeric glutathione S-transferase A1-1, we used heterodimers containing one wild-type (WT) active site and one active site with a single mutation at either Tyr9, Arg15, or Arg131. Tyr9 and Arg15 are part of the active site of the same subunit, while Arg131 contributes to the active site of the opposite subunit. The V max values of Tyr9 and Arg15 mutant enzymes were less than 2% that of WT, indicating their importance in catalysis. In contrast, V max values of Arg131 mutant enzymes were about 50−90% of that of WT enzyme while K m GSH values were approximately 3−8 times that of WT, suggesting that Arg131 plays a role in glutathione binding. The mutant enzyme (with a His6 tag) and the WT enzyme (without a His6 tag) were used to construct heterodimers (WT−Y9F, WT−Y9T, WT−R15Q, WT−R131M, WT−R131Q, and WT−R131E) by incubation of a mixture of wild-type and mutant enzyme at pH 7.5 in buffer containing 1,6-hexanediol, followed by dialysis against buffer lacking the organic solvent. The resultant heterodimers were separated from the wild-type and mutant homodimers using chromatography on nickel-nitrilotriacetic acid agarose. The V max values of all heterodimers were lower than expected for independent active sites. Our experiments demonstrate that mutation of an amino acid residue in one active site affects the activity in the other active site. Modeling studies show that key amino acid residues and water molecules connect the two active sites. This connectivity is responsible for the cross-talk between the active sites.