title: Hidden allostery in human glutathione transferase p1-1 unveiled by unnatural amino acid substitutions and inhibition studies.
abstract: Conventional steady-state kinetic studies of the dimeric human glutathione transferase (GST) P1-1 do not reveal obvious deviations from Michaelis-Menten behavior. By contrast, engineering of the key residue Y50 of the lock-and-key motif in the subunit interface reveals allosteric properties of the enzyme. The low-activity mutant Y50C, characterized by 150-fold decreased kcat and 300-fold increased KM(GSH) values, displays an apparent Hill coefficient of 0.82±0.22. Chemical alkylation of the sulfhydryl group of Y50C by unnatural n-butyl or n-pentyl substitutions enhances the catalytic efficiency kcat/KM(GSH) to near the wild-type value but still yields Hill coefficients of 0.61±0.08 and 0.86±0.1, respectively. Thus, allosteric kinetic behavior is not dependent on low activity of the enzyme. On the other hand, S-cyclobutylmethyl-substituted Y50C, which also displays high catalytic efficiency, has a Hill coefficient of 0.99±0.11, showing that subtle differences in structure at the subunit interface influence the complex kinetic behavior. Furthermore, inhibition studies of native GST P1-1 using ethacrynic acid demonstrate that a ligand bound noncovalently to the wild-type enzyme also can elicit allosteric kinetic behavior. Thus, we conclude that the GST P1-1 structure has intrinsic allostery that becomes overt under some, but not all, ambient conditions.
authors: Molecular Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, 12311 Cairo, Egypt.
authors: Usama M
authors: UM
authors: Hegazy
authors: Yaman
authors: Y
authors: Musdal
authors: Bengt
authors: B
authors: Mannervik
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics; D000495:Allosteric Site / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D051549:Glutathione S-Transferase pi / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D051549:Glutathione S-Transferase pi