title: Engineering a new C-terminal tail in the H-site of human glutathione transferase P1-1: structural and functional consequences.
abstract: We have sought the structural basis for the differing substrate specificities of human glutathione transferase P1-1 (class Pi) and human glutathione transferase A1-1 (class Alpha) by adding an extra helix (helix 9), found in the electrophilic substrate-binding site (H-site) of the human class Alpha enzyme, at the C terminus of the human class Pi enzyme. This class Pi-chimera (CODA) was expressed in Escherichia coli, purified and characterized by kinetic and crystallographic approaches. The presence of the newly engineered tail in the H-site of the human Pi enzyme alters its catalytic properties towards those exhibited by the human Alpha enzyme, as assessed using cumene hydroperoxide (diagnostic for class Alpha enzymes) and ethacrynic acid (diagnostic for class Pi) as co-substrates. There is a change of substrate selectivity in the latter case, as the k(cat)/K(m)(EA) value decreases about 70-fold, compared to that of class Pi. With 1-chloro-2,4-dinitrobenzene as co-substrate there is a loss of catalytic activity to about 2% with respect to that of the Pi enzyme. Crystallographic and kinetic studies of the class Pi-chimera provide important clues to explain these altered catalytic properties. The new helix forms many complimentary interactions with the rest of the protein and re-models the original electrophilic substrate-binding site towards one that is more enclosed, albeit flexible. Of particular note are the interactions between Glu205 of the new tail and the catalytic residues, Tyr7 and Tyr108, and the thiol moiety of glutathione (GSH). These interactions may provide an explanation of the more than one unit increase in the pK(a) value of the GSH thiolate and affect both the turnover number and GSH binding, using 1-chloro-2,4-dinitrobenzene as co-substrate. The data presented are consistent with the engineered tail adopting a highly mobile or disordered state in the apo form of the enzyme.
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica snc, 00133 Rome, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Micaloni
authors.forename: Geoffrey K-W
authors.initials: GK
authors.lastname: Kong
authors.forename: Anna P
authors.initials: AP
authors.lastname: Mazzetti
authors.forename: Marzia
authors.initials: M
authors.lastname: Nuccetelli
authors.forename: Giovanni
authors.initials: G
authors.lastname: Antonini
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Stella
authors.forename: William J
authors.initials: WJ
authors.lastname: McKinstry
authors.forename: Galina
authors.initials: G
authors.lastname: Polekhina
authors.forename: Jamie
authors.initials: J
authors.lastname: Rossjohn
authors.forename: Giorgio
authors.initials: G
authors.lastname: Federici
authors.forename: Giorgio
authors.initials: G
authors.lastname: Ricci
authors.forename: Michael W
authors.initials: MW
authors.lastname: Parker
authors.forename: Mario
authors.initials: M
authors.lastname: Lo Bello
mesh_terms: D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005978:Glutathione / Q000378:metabolism; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014783:Viscosity
chemical_list: D001051:Apoenzymes; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha; D005978:Glutathione