Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II of the cellular detoxification mechanism and are associated with increased susceptibility to cancer development and resistance to anticancer drugs. The present study aims to evaluate the ligandability of the human GSTM1-1 isoenzyme (hGSTM1-1) using a broad range of structurally diverse pesticides as probes. The results revealed that hGSTM1-1, compared to other classes of GSTs, displays limited ligandability and ligand-binding promiscuity, as revealed by kinetic inhibition studies. Among all tested pesticides, the carbamate insecticide pirimicarb was identified as the strongest inhibitor towards hGSTM1-1. Kinetic inhibition analysis showed that pirimicarb behaved as a mixed-type inhibitor toward glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). To shine a light on the restricted hGSTM1-1 ligand-binding promiscuity, the ligand-free crystal structure of hGSTM1-1 was determined by X-ray crystallography at 1.59 Å-resolution. Comparative analysis of ligand-free structure with the available ligand-bound structures allowed for the study of the enzyme’s plasticity and the induced-fit mechanism operated by hGSTM1-1. The results revealed important structural features of the H-site that contribute to xenobiotic-ligand binding and specificity. It was concluded that hGSTM1-1 interacts preferentially with one-ring aromatic compounds that bind at a discrete site which partially overlaps with the xenobiotic substrate binding site (H-site). The results of the study form a basis for the rational design of new drugs targeting hGSTM1-1.

The reactive adenosine derivative, adenosine 5′-O-[S-(4-hydroxy-2,3-dioxobutyl)]-thiophosphate (AMPS-HDB), contains a dicarbonyl group linked to the purine nucleotide at a position equivalent to the pyrophosphate region of NAD⁺. AMPS-HDB was used as a chemical label towards Candida boidinii formate dehydrogenase (CbFDH). AMPS-HDB reacts covalently with CbFDH, leading to complete inactivation of the enzyme activity. The inactivation kinetics of CbFDH fit the Kitz and Wilson model for time-dependent, irreversible inhibition (K_D = 0.66 ± 0.15 mM, first order maximum rate constant k₃ = 0.198 ± 0.06 min⁻¹). NAD⁺ and NADH protects CbFDH from inactivation by AMPS-HDB, showing the specificity of the reaction. Molecular modelling studies revealed Arg174 as a candidate residue able to be modified by the dicarbonyl group of AMPS-HDB. Arg174 is a strictly conserved residue among FDHs and is located at the Rossmann fold, the common mononucleotide-binding motif of dehydrogenases. Arg174 was replaced by Asn, using site-directed mutagenesis. The mutant enzyme CbFDHArg174Asn was showed to be resistant to inactivation by AMPS-HDB, confirming that the guanidinium group of Arg174 is the target for AMPS-HDB. The CbFDHArg174Asn mutant enzyme exhibited substantial reduced affinity for NAD⁺ and lower thermostability. The results of the study underline the pivotal and multifunctional role of Arg174 in catalysis, coenzyme binding and structural stability of CbFDH.