Abstract
Glutathione transferases (GSTs) are enzymes that play a critical role in cellular detoxication by catalyzing the nucleophilic attack of glutathione on the electrophilic center of a number of xenobiotic compounds, including many therapeutic drugs. Mutations of amino acid residues in the glutathione-binding site of human glutathione transferase P1–1, namely W39C, K45A, Q52A, Q52K, and Q52E, have been engineered. The recombinant mutant proteins were expressed in Escherichia coli, but only mutants K45A, Q52A, and Q52K showed measurable activity. Steady-state kinetics comparing glutathione with the alternative thiol substrate γ-glutamylcysteine demonstrated the importance of the glycine residue in glutathione for high catalytic efficiency. Inhibition experiments with a set of glutathione analogs structurally related to the therapeutic drugs Telintra and Telcyta enabled determination of binding energies that were contributed by different substituents. The effects of substituting amino acid side chains in the glutathione-binding site of the enzyme on binding the glutathione derivatives and catalysis were evaluated.
Highlights
Human glutathione transferase (GST) P1–1 is a member of a superfamily of detoxication enzymes, the GST superfamily [1,2]
Inhibition of the enzyme could, be expected to increase the efficiency of cytostatic drugs that, otherwise, would be inactivated by the enzyme [5]. This notion has been a rationale for the design of GST P1–1 inhibitors for use as adjuvant agents in chemotherapy [6,7]
The lysate had virtually no catalytic activity with CDNB, and the mutant enzyme could not be obtained as a pure protein
Summary
Human glutathione transferase (GST) P1–1 is a member of a superfamily of detoxication enzymes, the GST superfamily [1,2] This cytosolic enzyme is overexpressed in many malignant tumors [3,4]. Inhibition of the enzyme could, be expected to increase the efficiency of cytostatic drugs that, otherwise, would be inactivated by the enzyme [5]. This notion has been a rationale for the design of GST P1–1 inhibitors for use as adjuvant agents in chemotherapy [6,7]. Each subunit can bind a molecule of glutathione (GSH) and a hydrophobic electrophile in two adjacent sites of the catalytically active center, the G-site and the H-site, defined by Mannervik et al [8]
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