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 Cys 16 with Ala increases the rate of GSH release from the active site causing a 10-fold decrease of affinity toward GSH. Cys 16 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.

Mitochondrion as a Novel Site of Dichloroacetate Biotransformation by Glutathione Transferase ζ1

“…Cys16 was found to be particularly important in maintaining proper binding and orientation with GSH. Mutation of Cys16 to Ala caused dramatic increases in K m values for GSH with various substrates (Board et al, 2003;Ricci et al, 2004). As demonstrated in mouse GSTA4, the mitochondrial form is more heavily phosphorylated than the cytosolic form while possessing the same primary protein sequence (Robin et al, 2003).…”

The mercapturic acid pathway

“…Kinetic studies with hGSTZ1c-1c indicate that the activesite Cys16 is not a catalytic residue, although Cys16 is has an important role in binding of both GSH and electrophilic cosubstrates (Board et al 2003;Ricci et al 2004). The enzyme also shows half-site reactivity: binding of GSH to one subunit effectively silences the second subunit as it is unable to bind and activate GSH (Ricci et al 2004).…”

Disulfide bond formation protects Arabidopsis thaliana glutathione transferase tau 23 from oxidative damage

“…Since activation parameters are the difference in energy from the activated E:S # complex and the E:S ground state, we sought to investigate the energy profile of the E:S complex formation. It has been demonstrated that GST enzymes catalysing the conjugation of GSH and CDNB are under a rapid-equilibrium random mechanism [42][43][44][45][46]. Assuming that GSTU23 is also under a rapid-equilibrium mechanism, K m can be considered as K D and a ΔG° for the E:S complex formation can be calculated [47].…”