Programmed Delivery of Novel Functional Groups to the Alpha Class Glutathione Transferases

Abstract

Here we describe a new route to site- and class-specific protein modification that will allow us to create novel functional proteins with artificial chemical groups. Glutathione transferases from the alpha but not the mu, pi, omega, or theta classes can be rapidly and site-specifically acylated with thioesters of glutathione (GS-thioesters) that are similar to compounds that have been demonstrated to occur in vivo. The human isoforms A1-1, A2-2, A3-3, and A4-4 from the alpha class all react with the reagent at a conserved tyrosine residue (Y9) that is crucial in catalysis of detoxication reactions. The yield of modified protein is virtually quantitative in less than 30 min under optimized conditions. The acylated product is stable for more than 24 h at pH 7 and 25 degrees C. The modification is reversible in the presence of excess glutathione, but the labeled protein can be protected by adding S-methylglutathione. The stability of the ester with respect to added glutathione depends on the acyl moiety. The reaction can also take place in Escherichia coli lysates doped with alpha class glutathione transferases. A control substance that lacks the peptidyl backbone required for binding to the glutathione transferases acylates surface-exposed lysines. There is some acyl group specificity since one out of the three different GS-thioesters that we tried was not able to acylate Y9.

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

“…After the standard 5 h of incubation, the yield is quantitative in the GS-4 reaction but the GSB reaction has only reached about 70% completion. The yield can thus most likely be fine-tuned since this was possible in the case of GSB and hGST A1-1 where the degree of modification was a function of both time and concentration of reagent (22).…”

“…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). All tripeptide reagents were analyzed with reversedphase HPLC using a C 8 column and a shallow H 2 O:ACN (both 0.1% TFA) gradient.…”

Incorporation of a single His residue by rational design enables thiol-ester hydrolysis by human glutathione transferase A1-1

“…GSH was prepared by solid-phase peptide synthesis using standard 9-fluorenylmethoxycarbonyl (Fmoc) protocols and Fmoc-Gly-Wang resin (0.7-mmol scale) as described (29). Cys was orthogonally protected with a 4-methoxytrityl group, and the thiol was selectively deprotected with 1% trifluoroacetic acid in dichloromethane to enable thiol-ester coupling with benzoic acid.…”

“…Global deprotection and cleavage from the solid support was enabled by trifluoroacetic acid͞H 2 O (97.5:2.5 dilution), followed by precipitation of the peptide with diethyl ether. GSB was purified by reverse-phase HPLC (29). GSB concentrations were determined spectrophotometrically by using an extinction coefficient of 266 ϭ 7,889 M Ϫ1 ⅐cm Ϫ1 .…”

“…The fate of the benzoyl group in the wild-type protein was determined by matrix-assisted laser desorption ionization-time of flight MS by subjecting the reaction mixture to cleavage by trypsin or S. aureus protease V8 and comparing the molecular masses of the peptide fragments to those obtained in the absence of substrate. The fragment containing Y9 (residues 7-13 for trypsin) was readily observed in the wild-type enzyme and was acylated to a large degree by benzoic acid under reaction conditions (29). Under the same conditions, A216H showed only minor acylation at the side chain of Y9.…”