Identification of Covalent Modifications Derived from the GSTZ1‐catalyzed Metabolism of Dichloroacetate

Abstract

Glutathione transferase zeta 1 (GSTZ1) is a cytosolic and mitochondrial enzyme with a critical role in tyrosine catabolism via its trans‐isomerization of maleylacetoacetate (MAA) and maleylacetone (MA). Dichloroacetate (DCA) is an investigational drug utilized in the treatment of acquired and congenital mitochondrial diseases. Dechlorination of DCA to its inactive metabolite, glyoxylate, is catalyzed by GSTZ1, and allelic variants within GSTZ1 affect the kinetics of DCA conversion. DCA is a mechanism‐based inactivator of GSTZ1, and chronic DCA dosing results in the auto‐inhibition of DCA metabolism. The tissue accumulation of DCA, MAA, and MA due to GSTZ1 degradation may contribute to the observed side effects of DCA, as chronic dosing can lead to the development of reversible peripheral neuropathy in some patient populations. The mechanism for DCA‐induced inactivation of GSTZ1 is postulated to proceed via the formation of a carbonium‐sulfonium intermediate derived from the GSTZ1‐catalyzed reaction of glutathione and DCA. The intermediate may react with nucleophilic sites on GSTZ1 to yield covalently‐modified enzyme or undergo hydrolysis to form glyoxylate. Modification to Cys‐16 within the 14SSCSWR19 glutathione binding motif of GSTZ1 has been described as a mechanism of DCA inactivation, although the mechanism for haplotype differences remain unclear. Therefore, we examined multiple GSTZ1 isoforms for variations in covalent modifications derived from DCA metabolism. GSTZ1 haplotypes 1B, 1C, and 1D (0.5 mg, each) were incubated for 10 hours at 37C in the presence of 2 mM DCA, 5 mM GSH, and 1 mg/mL BSA as stabilizer. The precipitates were collected by centrifugation and separated via SDS‐PAGE to isolate bands for GSTZ1, which were then excised and subjected to tryptic digestion for mass spectral characterization. We confirmed the addition of glyoxylate (+74 m/z) to multiple serine residues within GSTZ1 and present the following serine modifications: Ser‐47 (1C), Ser‐187 (1B and 1D), Ser‐188 (1C and 1D), and Ser‐202 (1B, 1C, and 1D). Each haplotype demonstrated a single adduct of +364 m/z to Cys‐205 within the peptide 193LLVLEAFQVSHPCR206. The change in mass is consistent with an adduct that contains a molecule of glutathione bound to the backbone of DCA, which we describe as S‐carboxy‐methylglutathione. This is a newly characterized modification for the DCA‐induced inactivation of GSTZ1. The incidence of S‐carboxy‐methylglutathione was low and its role in GSTZ1 inactivation has yet to be explored. DCA‐induced inactivation of GSTZ1 could be due, in part, to the aggregation of proteins covalently modified by glyoxylate, and the number of sites accessible to covalent linkages may vary between GSTZ1 haplotypes.Support or Funding InformationSupported in part by the US Public Health Service GM 099871This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.