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Identification of non‐substrate amino acid analogs that alter activity and substrate binding in gamma‐glutamylcysteine
Author(s) -
Boettcher Colin,
Rozenboom Kelly,
Kelly Brenda S.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.676.7
Subject(s) - cysteine , substrate (aquarium) , enzyme , biochemistry , binding site , amino acid , active site , mutagenesis , glutathione , chemistry , glycine , stereochemistry , biology , mutation , gene , ecology
The enzyme, gamma‐glutamylcysteine ligase (gamma‐GCL) catalyzes the first and rate‐limiting step in the synthesis of glutathione, an important detoxicant in nearly all eukaryotes that has been implicated in chemotherapeutic resistance. In the present study, non‐substrate amino acid analogs that activate or inhibit activity of E. coli gamma‐GCL in a concentration‐dependent manner were identified. Fluorescence titration studies confirmed that the analogs specifically bind to the enzyme and alter the binding affinity of the biological substrate cysteine or a cysteine‐mimic. Moreover, sigmoidal binding curves suggest that the enzyme exhibits at least two distinct, cooperative binding sites that may be filled with a cysteine substrate or non‐substrate analog. We hypothesize that E. coli gamma‐GCL is monomeric and has an active site that, when bound with a non‐substrate analog, significantly impacts whether cysteine can bind in a productive (activating) or non‐productive (inhibiting) manner. Current and future studies include confirmation of the oligomeric state of the enzyme, identification of structural characteristics that result in high affinity binding, and site‐directed mutagenesis of proposed active site residues in order to gain further insight into the structure of E. coli gamma‐GCL. This study is supported by Sigma Xi and the Langsjoen fund at Gustavus Adolphus College.