Combinatorial Chemical Reengineering of the Alpha Class Glutathione Transferases

Abstract

Previously, we discovered that human glutathione transferases (hGSTs) from the alpha class can be rapidly and quantitatively modified on a single tyrosine residue (Y9) using thioesters of glutathione (GS-thioesters) as acylating reagents. The current work was aimed at exploring the potential of this site-directed acylation using a combinatorial approach, and for this purpose a panel of 17 GS-thioesters were synthesized in parallel and used in screening experiments with the isoforms hGSTs A1-1, A2-2, A3-3, and A4-4. Through analytical HPLC and MALDI-MS experiments, we found that between 70 and 80% of the reagents are accepted and this is thus a very versatile reaction. The range of ligands that can be used to covalently reprogram these proteins is now expanded to include functionalities such as fluorescent groups, a photochemical probe, and an aldehyde as a handle for further chemical derivatization. This site-specific modification reaction thus allows us to create novel functional proteins with a great variety of artificial chemical groups in order to, for example, specifically tag GSTs in biological samples or create novel enzymatic function using appropriate GS-thioesters.

Ligand-Directed Labeling of a Single Lysine Residue in hGST A1-1 Mutants

“…The logP values span a range from 1.3 (4-acetamidobenzoic acid) to 3.8 (2-phenyl-4-quinolinecarboxylic acid) and only the acids with a logP value below 2.3 are able to label K216 ( Table 4). The exception is GS-9 even though it has a logP of 2.2, but this is not surprising as discussed above (23). This means that K216 cannot be modified with di-orthosubstituted or very hydrophobic GS-thiolesters, presumably due to the hydrophilic character of the lysine sidechain.…”

“…In the case of GSB, we have thus increased the stability of the protein conjugate at the expense of the speed of the reaction. However, the rate is highly reagent-dependent (23). c + and -corresponds to modified or not modified K216 fragment.…”

“…The product is unstable toward GSH (22), and addition of a fresh batch of another GSthiolester to premodified protein results in scrambling of the acyl groups (23). Even though the benzoic acidderived Y9-ester is stable, for other Y9-esters this bond is slowly hydrolyzed (23), and with this in mind we designed and prepared two lysine mutants of hGST A1-1, Y9K and A216K, that we hoped would form stable amide bonds with the acyl groups of the reagents (Scheme 2). The Y9K mutant is a straightforward change to see whether the targeted tyrosine could be directly substituted by a lysine.…”

“…The UV measurements were performed using a Varian Cary 100 Scan UV-Visible spectrophotometer and analyzed with the CaryWin UV software. The syntheses of the reagents have been described before, and we used the stock solutions described in the previous report (23). The synthesis of the substrate GSB has also been described previously (22).…”

Irreversibly binding anti-metal chelate antibodies: Artificial receptors for pretargeting

“…Others have employed proximity-accelerated alkylation to label nucleophilic amino acid residues located proximal to binding sites rather than targeting nucleophilic residues in the active sites of proteins [75,76,[84][85][86][87][88]. While some have labeled naturally occurring sites, the kinetics for the reaction have been slow [84,86].…”