- Alkylation and inactivation of human glutathione transferase zeta (hGSTZ1-1) by maleylacetone and fumarylacetone
-
Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate or maleylacetone (MA) to fumarylacetoacetate or fumarylacetone (FA), respectively. GSTZ1-1 also catalyzes the glutathione-dependent biotransformation of a range of α-haloacids, including dichloroacetic acid. The objective of this study was to investigate the mechanism of inactivation of hGSTZ1-1 by MA and FA and to determine the covalent modification of hGSTZ1-1 by MA and FA in the presence and absence of glutathione. MA and FA (0.01-1 mM) inactivated all hGSTZ1-1 polymorphic variants in a concentration- and time-dependent manner, and this inactivation was blocked by glutathione. The C16A mutant of hGSTZ1c-1c was partially inactivated by MA and FA. Electrospray ionization-tandem mass spectrometry and SALSA (Scoring Algorithm for Spectral Analysis) analyses of tryptic digests of hGSTZ1 polymorphic variants revealed that the active site (SSC*SWR) and C-terminal (LLVLEAFQVSHPC*R) cysteine residues of hGSTZ1-1 were covalently modified by MA and FA. MA and FA adduction resulted in diagnostic 156-Da shifts in the masses of the modified peptide ions and in their MS-MS fragment ions. Alkylation of the active-site cysteine residues, but not of the C-terminal cysteine, was relatively less intense when hGSTZ1-1 polymorphic variants were incubated with MA or FA in the presence of S-methyl glutathione. These data indicate that MA and FA are substrate and product inactivators of hGSTZ1-1 and covalently modify hGSTZ1-1 at the active-site cysteine residue in the absence of glutathione. The observation that inactivation was blocked by glutathione indicates that binding of glutathione to the active site prevents reaction of MA or FA with the active-site cysteine residue. These data also indicate that MA and FA may covalently modify and inactivate other proteins that have accessible cysteine residues and may, thereby, contribute to dichloroacetic acid-induced or hypertyrosinemia type-I-associated toxicities.
- Lantum, Hoffman B. M.,Liebler, Daniel C.,Board, Philip G.,Anders
-
-
Read Online
- Kinetics of the biotransformation of maleylacetone and chlorofluoroacetic acid by polymorphic variants of human glutathione transferase zeta (hGSTZ1-1)
-
Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate and the biotransformation of a range of α-haloacids. The objective of this study was to determine the kinetics of the biotransformation of maleylacetone (MA), an analogue of the natural substrate maleylacetoacetate, and chlorofluoroacetic acid (CFA) by polymorphic variants of recombinant hGSTZ1-1. The kcat of the four variants of hGSTZ1-1 with MA as the substrate followed the order: 1c-1c > 1b-1b > 1d-1d > 1a-1a whereas the kcat for the biotransformation of CFA followed the order: 1a-1a > 1b-1b ~ 1c-1c ~ 1d-1d. The turnover rates of MA were much higher than those of CFA for each variant and ranged from 22-fold (1a-1a) to 980-fold differences (1c-1c). The catalytic efficiencies of hGSTZ1-1 variants with MA as the substrate were much greater than those with CFA as the substrate, but little difference among the polymorphic variants was observed. MA was a mixed inhibitor of all variants with CFA as substrate: the mean competitive inhibition constant (KicMA) for all variants was about 100 μM and the mean uncompetitiv- inhibition constant (KiuMA) was about 201 μM. Hence, MA and α-haloacids apparently compete for the same active site on the enzyme. DCA-induced inactivation of the four variants showed that the inactivated enzymes show markedly reduced isomerase activities. The residual activities were different for each variant: 1a-1a (12%) > 1b-1b ~ 1c-1c ~ 1d-1d (5%). This is the first kinetic analysis of polymorphic variants of hGSTZ1-1, and the similarity of the kinetic constants for hGSTZ1-1 variants with either MA or CFA as substrates indicates that few differences in DCA-induced perturbations of tyrosine metabolism would likely be observed in humans.
- Lantum, Hoffman B. M.,Board, Philip G.,Anders
-
-
Read Online