title: Cooperative binding of gamma-glutamyl substrate to human glutathione synthetase.
abstract: Human glutathione synthetase is responsible for catalyzing the final step in glutathione biosynthesis. It is a homodimer with a monomer subunit MW of 52 kDa. Kinetic analysis reveals a departure from linearity of the Lineweaver-Burk double reciprocal plot for the binding of gamma-glutamyl substrate, indicating cooperative binding. The measured apparent K(m) values for gamma-glutamyl-alpha-aminobutyrate (an analog of gamma-glutamyl-alpha-aminobutyrate) are 63 and 164 microM, respectively. Neither ATP (K(m) of 248 microM) nor glycine (K(m) of 452 microM) exhibits such cooperative binding behavior. Although ATP is proposed to play a key role in the sequential binding of gamma-glutamyl substrate to the enzyme, the cooperative binding of the gamma-glutamyl substrate is not affected by alterations of ATP concentration. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0.75, indicating negative cooperativity. Our studies, for the first time, show that human glutathione synthetase is an allosteric enzyme with cooperative binding for gamma-glutamyl substrate.
authors.affiliation: Department of Pediatrics, Karolinska Institute, Huddinge University Hospital, 141 86 Huddinge, Sweden.
authors.forename: R
authors.initials: R
authors.lastname: Njalsson
authors.forename: S
authors.initials: S
authors.lastname: Norgren
authors.forename: A
authors.initials: A
authors.lastname: Larsson
authors.forename: C S
authors.initials: CS
authors.lastname: Huang
authors.forename: M E
authors.initials: ME
authors.lastname: Anderson
authors.forename: J L
authors.initials: JL
authors.lastname: Luo
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D004151:Dipeptides / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005981:Glutathione Synthase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004151:Dipeptides; D011994:Recombinant Proteins; C042262:gamma-glutamyl-alpha-aminobutyrate; D000255:Adenosine Triphosphate; D005981:Glutathione Synthase; D005978:Glutathione