Kinetics of the Biotransformation of Maleylacetone and Chlorofluoroacetic Acid by Polymorphic Variants of Human Glutathione Transferase Zeta (hGSTZ1-1)

Abstract

Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate and the biotransformation of a range of alpha-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 k(cat) 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 k(cat) for the biotransformation of CFA followed the order: 1a-1a > 1b-1b approximately 1c-1c approximately 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 (K(ic)(MA)) for all variants was about 100 microM, and the mean uncompetitive inhibition constant (K(iu)(MA)) was about 201 microM. Hence, MA and alpha-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 approximately 1c-1c approximately 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.

The mercapturic acid pathway

“…The rate constants (k inact ) for the GSH-dependent, DCA-induced inactivation of hGSTZ1-1 variants are similar for hGSTZ1b-1b, -1c-1c, and -1d-1d ($7 Â 10 À2 min À1 ), which are higher than that of hGSTZ1a-1a (3 Â 10 À2 min À1 ) (Tzeng et al 2000). With recombinant hGSTZ1-1, the rate constants (k cat /K m ) for the biotransformation of maleylacetone (134, Figure 11) followed the order: 1d-1d ¼ 1 b-1b > 1c-1c > 1a-1a, whereas the rate constants for the biotransformation of chlorofluoroacetic acid were similar among the four allelic variants (Lantum et al 2002b). In children given DCA (132, Figure 11) for management of congenital mitochondrial disorders, the plasma drug t1 =2 was shorter in hGSTZ1c-1c (EGT, see above) carriers compared with hGSTZ1c-1c noncarriers (Shroads et al 2015).…”

“…As indicated above, GSTZ1-1 fails to catalyze the conjugation of typical GST substrates or catalyzes the reactions at low rates (Board et al 1997). It was later shown that substrates for GSTZ1-1 include DCA (132, Figure 11) and a range of a-haloalkanoic acids as well as maleylacetone (134, Figure 11) (Tong et al 1998b;Lantum et al 2002b). hGSTZ1-1 catalyzes the reduction of organic hydroperoxides, e.g.…”

Therapeutic applications of dichloroacetate and the role of glutathione transferase zeta-1

“…The role of GSTZ1 in isomerization of maleylacetoacetate and maleylacetone to fumarylacetoacetate and fumarylacetone is thought to be important in reducing the potential toxicity of these electrophilic, chemically reactive alpha-beta-unsaturated ketones. If maleylacetoacetate and maleylacetone are not isomerized, these intermediates could form covalent bonds with cellular nucleophiles such as free SH, NH 2 or OH groups found in proteins from the amino acids cysteine, lysine, arginine, serine and threonine, or with NH 2 groups in DNA bases through Michael addition reactions, as shown in Figure 6: evidence for maleylacetone adducts with protein cysteine residues has been presented (Lantum, et al, 2002b). If critical proteins or sites in DNA form covalent adducts, this can alter their structure and lead to toxicity (Guengerich & MacDonald, 2007).…”

Clarification of the role of key active site residues of glutathione transferase Zeta/maleylacetoacetate isomerase by a new spectrophotometric technique

“…Despite this limitation, the assay can be readily used to quantify the activity of purified GSTZ1-1. The optimal activity of hGSTZ1c-1c was relatively broad but had a peak at pH 10.5, which is considerably higher than the optimum with CFA as a substrate (pH 7.8-8.2) [25]; most other GSTs appear to have optima around pH 8.5 [13]. It was notable that among the naturally occurring allelic variants of hGSTZ1-1, we found that hGSTZ1a-1a had significantly higher activity with BNPP than the other variants.…”