title: Binding and kinetic mechanisms of the Zeta class glutathione transferase.
abstract: The Zeta class of glutathione transferases (GSTs) has only recently been discovered and hence has been poorly characterized. Here we investigate the substrate binding and kinetic mechanisms of the human Zeta class GSTZ1c-1c by means of pre-steady state and steady-state experiments and site-directed mutagenesis. Binding of GSH occurs at a very low rate compared with that observed for the more recently evolved GSTs (Alpha, Mu, and Pi classes). Moreover, the single step binding mechanism observed in this enzyme is reminiscent of that found for the Theta class enzyme, whereas the Alpha, Mu, and Pi classes have adopted a multistep binding mechanism. Replacement of Cys16 with Ala increases the rate of GSH release from the active site causing a 10-fold decrease of affinity toward GSH. Cys16 also plays a crucial role in co-substrate binding; the mutant enzyme is unable to bind the carcinogenic substrate dichloroacetic acid in the absence of GSH. However, both substrate binding and GSH activation are not rate-limiting in catalysis. A peculiarity of the hGSTZ1c-1c is the half-site activation of bound GSH. This suggests a primitive monomer-monomer interaction that, in the recently diverged GSTP1-1, gives rise to a sophisticated cooperative mechanism that preserves the catalytic efficiency of this GST under stress conditions.
authors.affiliation: Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, 00133 Rome, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Ricci
authors.forename: Paola
authors.initials: P
authors.lastname: Turella
authors.forename: Francesca
authors.initials: F
authors.lastname: De Maria
authors.forename: Giovanni
authors.initials: G
authors.lastname: Antonini
authors.forename: Luisa
authors.initials: L
authors.lastname: Nardocci
authors.forename: Philip G
authors.initials: PG
authors.lastname: Board
authors.forename: Michael W
authors.initials: MW
authors.lastname: Parker
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Carbonelli
authors.forename: Giorgio
authors.initials: G
authors.lastname: Federici
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Caccuri
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003999:Dichloroacetic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D003999:Dichloroacetic Acid; C407705:GSTZ1 protein, human; D005982:Glutathione Transferase; D005978:Glutathione