[Study on the interaction between the skin detoxifying enzyme glutathione S-transferase and the substances listed in the CE/39/2000 rules with the "skin" annotation, finalized to the biological monitoring of exposed subjects]. / Studio dell'interazione fra l'enzima detossificante cutaneo glutatione S-trasferasi e le sostanze elencate nella normativa CEE/39/2000 con notazione 'skin' finalizzato al monitoraggio biologico di soggetti esposti.

The interaction among chemicals listed in the Directive CE/39/2000 with skin notation and glutathione S-transferase (GSTP1-1) was studied by following two different experimental approaches. The compounds were incubated with the purified GST isoenzyme GSTP1-1 as well as with the human keratinocytes (PR5) selectively expressing GSTP1-1. Some of the molecules affected the enzymatic activity of both the purified and the intracellular GSTP1-1. In particular, 1,2-dichlorobenzene (DCB), ethylbenzene (ETB), cumene, Sulphotep and 2-eptanone (2-EPT) behaved as inhibitors of the purified GSTP1-1 enzyme, with different inhibition properties according to molecular structure. With the exception of Sulphotep showing a Ki value of 0.2 mM, all compounds reported above were characterized by high Ki values (between 2 and 16 mM) and therefore by low affinity towards GSTP1-1. These results make unlikely the use of a biosensor, based on immobilized GSTP1-1, for the detection of these molecules. On the contrary, Sulphotep can be the object of future investigations. It has to be stressed that the above listed compounds were effective on human keratinocytes, at concentrations two order of magnitude lower than that effective on purified GSTP1-1. In particular, cumene and DCB triggered a clear increase of the intracellular GSTP1-1 activity at concentrations lower than 0.1mM. These interesting results let to hypothesize the use of GSTP1-1 present in the keratinocytes as a marker for biological monitoring of workers exposed to these compounds as well as to evaluate the skin permeability of toxic compounds, not yet identified with a skin notation.

Potent isozyme-selective inhibition of human glutathione S-transferase A1-1 by a novel glutathione S-conjugate.

Elevated levels of glutathione S-transferases (GSTs) are among the factors associated with an increased resistance of tumors to a variety of antineoplastic drugs. Hence a major advancement to overcome GST-mediated detoxification of antineoplastic drugs is the development of GST inhibitors. Two such agents have been synthesized and tested on the human Alpha, Mu and Pi GST classes, which are the most representative targets for inhibitor design. The novel fluorescent glutathione S-conjugate L-gamma-glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (4) has been found to be a highly potent inhibitor of human GSTA1-1 in vitro (IC50=0.11+/-0.01 microM). The peptide is also able to inhibit GSTP1-1 and GSTM2-2 isoenzymes efficiently. The backbone-modified analog L-gamma-(gamma-oxa)glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (6), containing an urethanic junction as isosteric replacement of the gamma-glutamyl-cysteine peptide bond, has been developed as gamma-glutamyl transpeptidase-resistant mimic of 4 and evaluated in the same inhibition tests. The pseudopeptide 6 was shown to inhibit the GSTA1-1 protein, albeit to a lesser extent than the lead compound, with no effect on the activity of the isoenzymes belonging to the Mu and Pi classes. The comparative loss in biological activity consequent to the isosteric change confirms that the gamma-glutamyl moiety plays an important role in modulating the affinity of the ligands addressed to interact with GSH-dependent proteins. The new specific inhibitors may have a potential in counteracting tumor-protective effects depending upon GSTA1-1 activity.

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of substrate binding to the active site of glutathione transferases.

Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK(a) = 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k(on) and k(off) values at least hundred times lower (k(on) = (2.7 +/- 0.8) x 10(4) M(-1) s(-1), k(off) = 36 +/- 9 s(-1), at 37 degrees C). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK(a) value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results.

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of the catalytic activity of glutathione transferases.

Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate. This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k(cat) and k(cat)/K(m)((GSH)) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.