title: Tyrosine 50 at the subunit interface of dimeric human glutathione transferase P1-1 is a structural key residue for modulating protein stability and catalytic function.
abstract: The dimer interface in human GSTP1-1 has been altered by site-directed mutagenesis of Tyr50. It is shown that the effects of some mutations of this single amino acid residue are as detrimental for enzyme function as mutations of Tyr8 in the active site. The dimeric structure is a common feature of the soluble glutathione transferases and the structural lock-and-key motif contributing to the subunit-subunit interface is well conserved in classes alpha, mu, and pi. The key residue Tyr50 in GSTP1-1 was replaced with 5 different amino acids with divergent properties and the mutant proteins expressed and characterized. Mutant Y50F is an improved variant, with higher thermal stability and higher catalytic efficiency than the wild-type enzyme. The other mutants studied are also dimeric proteins, but have lower stabilities and catalytic activities that are reduced by a factor of 10(2)-10(4) from the wild-type value. Mutants Y50L and Y50T are characterized by a markedly increased K(M) value for GSH, while the effect is mainly due to decreased k(cat) values for mutants Y50A and Y50R. In conclusion, residue 50 in the interface governs both structural stability and catalytic function.
authors.affiliation: Department of Biochemistry, Biomedical Center, Uppsala University, Uppsala, SE-751 23, Sweden. gun.stenberg@biokem.uu.se
authors.forename: G
authors.initials: G
authors.lastname: Stenberg
authors.forename: A M
authors.initials: AM
authors.lastname: Abdalla
authors.forename: B
authors.initials: B
authors.lastname: Mannervik
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D002384:Catalysis; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000737:chemistry*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D013696:Temperature; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D007527:Isoenzymes; D014443:Tyrosine; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase