title: Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism.
abstract: Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k(cat) and k(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.
authors.affiliation: Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden
authors.forename: Lisa O
authors.initials: LO
authors.lastname: Nilsson
authors.forename: Maryam
authors.initials: M
authors.lastname: Edalat
authors.forename: Pär L
authors.initials: PL
authors.lastname: Pettersson
authors.forename: Bengt
authors.initials: B
authors.lastname: Mannervik
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D004137:Dinitrochlorobenzene / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D014783:Viscosity
chemical_list: D004137:Dinitrochlorobenzene; D007527:Isoenzymes; D005982:Glutathione Transferase; D005978:Glutathione