Evaluation of the role of two conserved active-site residues in Beta class glutathione S-transferases

Glutathione S-transferases (GSTs) normally use hydroxy-group-containing residues in the N-terminal domain of the enzyme for stabilizing the activated form of the co-substrate, glutathione. However, previous mutagenesis studies have shown that this is not true for Beta class GSTs and thus the origin of the stabilization remains a mystery. The recently determined crystal structure of Proteus mirabilis GST B1-1 (PmGST B1-1) suggested that the stabilizing role might be fulfilled in Beta class GSTs by one or more residues in the C-terminal domain of the enzyme. To test this hypothesis we mutated His¹⁰⁶ and Lys¹⁰⁷ of PmGST B1-1 to investigate their possible role in the enzyme's catalytic activity. His¹⁰⁶ was mutated to Ala, Asn and Phe, and Lys¹⁰⁷ to Ala and Arg. The effects of the replacement on the activity, thermal stability and antibiotic-binding capacity of the enzyme were examined. The results are consistent with the involvement of His¹⁰⁶ and Lys¹⁰⁷ in interacting with glutathione at the active site but these residues do not contribute significantly to catalysis, folding or antibiotic binding.