Interaction of 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-t) with the acyl-CoA: Amino acid N-acyltransferase enzymes of bovine liver mitochondria
Abstract
The amino acid conjugation of the phenoxyherbicides 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T) by animals was examined at the level of the enzymes catalyzing the reactions. The phenoxyherbicides were not substrates for the bile acid conjugating system but were substrates for the mitochondrial xenobiotic conjugating system. The two mitochondrial xenobiotic-CoA : amino acid N-acyltransferases (benzoyltransferase and phenylacetyltransferase) were separated and tested for activity towards 2,4-D-CoA and 2,4,5-T-CoA. The phenylacetyltransferase showed activity towards phenylacetyl-CoA, phenoxyacetyl-CoA and 2,4-D-CoA, but not 2,4,5-T-CoA. Benzoyltransferase conjugated both 2,4-D-CoA and 2,4,5-T-CoA. The overall rates of conjugation of the phenoxyherbicides were slow relative to the standard substrates with both enzymes. This slow rate was found to be due in both cases to a relatively high Km for glycine, and a very slow catalytic rate constant. Both enzymes did, however, have a very high affinity for 2,4-D-CoA and 2,4,5-T-CoA so these compounds proved to be potent alternate substrate inhibitors of both enzymes. The data show that the inefficient in vivo conjugation of the phenoxyherbicides relative to structurally similar compounds can be understood in terms of the kinetic properties of the mitochondrial N-acyltransferases. Further, the potential for the interference of the phenoxyherbicides with the conjugation of other compounds is revealed.
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Cited by (22)
Enzymology of Amino Acid Conjugation Reactions
2018, Comprehensive Toxicology: Third EditionAmino acid conjugation is a route of metabolism for various xenobiotic carboxylic acids and bile acids; however, knowledge of the enzymology (e.g., multiplicity of enzymes, substrate selectivity, catalytic mechanism, and protein structure), polymorphisms, and gene regulation is not as advanced as the cytochromes P450 and uridine 5′-diphosphate (UDP)-glucuronosyltransferases. Mechanistically, amino acid conjugation is a coupled enzyme system involving a mitochondrial adenosine triphosphate (ATP)-dependent acid medium-chain acyl-CoA synthetase (ACSM), which catalyzes the formation of an acyl-CoA thioester, and an acyl-CoA:glycine N-acyltransferase (GLYAT) that links the acyl group to glycine. Conjugation of bile acids is extramitochondrial involving initially enzymes located in the endoplasmic reticulum, while conjugation of bile acid-CoAs involves a peroxisomal bile acid-CoA:amino acid N-acyltransferase (BAAT). Within mitochondria, amino acid conjugation terminates the biological reactivity of xenobiotic acyl-CoAs, whereas conjugation of bile acids increases aqueous solubility and promotes bile acid flow and intestinal absorption. For both pathways, the amino acid utilized is species dependent. In mammals, excretion is predominantly as either glycine or taurine conjugates. Amino acid conjugation is important in both health and disease. Manipulation of mitochondrial amino acid conjugation is used in the treatment of nonketotic hyperglycinemia and hyperammonemia, and failure to conjugate bile acids can lead to cholestasis and loss of absorption of fat and fat-soluble vitamins. This chapter reviews the enzymology, substrate and inhibitor profile, and factors that regulate the activity of ACSM, GLYAT, and BAAT.
Enzymology of Amino Acid Conjugation Reactions
2010, Comprehensive Toxicology, Second EditionAmino acid conjugation is a route of metabolism for both a variety of xenobiotic carboxylic acids and also bile acids. However, knowledge of the overall enzymology (including multiplicity of enzymes, substrate selectivity, catalytic mechanism, protein structure, polymorphisms, and gene regulation) is not as advanced in comparison to the cytochromes P450 and uridine 5′-diphosphate (UDP)-glucuronosyltransferases. Amino acid conjugation differs mechanistically from other conjugating reactions in that it is a coupled enzyme system involving any one of a number of adenosine triphosphate (ATP)-dependent acid: CoA ligases and acyl-CoA:amino acid N-acyltransferases. In relation to xenobiotic carboxylic acids, amino acid conjugation involves enzymes located in the mitochondrial matrix while conjugation of bile acids is extramitochondrial involving enzymes located in the endoplasmic reticulum and peroxisomes. The first step in both xenobiotic and bile acid conjugation involves activation to the corresponding acyl-CoA, which is then a substrate for amino acid conjugation. Within mitochondria, amino acid conjugation terminates the biological reactivity of xenobiotic acyl-CoAs, whereas conjugation of bile acids increases aqueous solubility and promotes bile acid flow and intestinal absorption. For both pathways the amino acid utilized is species dependent and in mammals excretion is predominantly as either glycine or taurine conjugates. Amino acid conjugation has a fundamentally important role both in health and disease. Manipulation of mitochondrial amino acid conjugation is used in the treatment of nonketotic hyperglycinemia and hyperammonemia as failure to amino acid conjugate bile acids can lead to cholestasis and loss of absorption of fat and fat-soluble vitamins.
Developmental profile of mitochondrial glycine N-acyltransferase in human liver
1997, Journal of PediatricsObjective: To study the developmental profile of glycine N -acyltransferase (GAT) in the livers of children of various ages and to compare the total and specific GAT activity with that of the adult control subjects.
Methods: We measured the specific and the total mitochondrial activity of GAT in liver samples taken from 13 children 4 hours to 11 years of age. The samples were compared with those of control adults aged 24 to 40 years. Samples, either from liver-transplant donors or from autopsy, from those who died of a disorder not related to the liver, were obtained between 6 and 36 hours after death.
Results: At 4 hours after birth, very low specific activity and the total liver mitochondrial activity were observed (0.19 μmol/min per milligram protein and 210 mmol/min), with a steady increase up to age 7 months (2.51 μmol/min per milligram protein and 812 mmol/min). The mean specific and total GAT activity in children (n = 5) aged 18 months to 11 years was 6.38 ± 0.13 and 1389 ± 43 and in control adults aged 24 to 40 years (n = 3) was 6.5 ± 0.3 and 1461 ± 71 μmol/min per milligram protein and μmol/min, respectively. These specific and total GAT activity values from children aged 18 months to 11 years were not statistically significant (by analysis of variance and Mann-Whitney test) in comparison with the corresponding activity values from the adult control subjects.
Conclusions: Our results indicate that up to age 7 months, children have only 5% to 40% of liver GAT-specific activity, whereas the peak activity is achieved at 18 months and remains constant until age 40 years. The delayed development of GAT in children may thus compromise the detoxification of various drugs and xenobiotics. (J Pediatr 1997;130:1003-7)
In vitro evidence for involvement of CoA thioesters in peroxisome proliferation and hypolipidaemia
1994, BBA - Molecular Cell ResearchThe mechanisms of peroxisomal induction and hypolipidaemia caused by treatment with peroxisome proliferators, such as nafenopin and clofibrate, remain to be elucidated. Proposed mechanisms include receptor-mediated processes or adaptations resulting from disruption of hepatic lipid metabolism. The latter mechanism was investigated in a series of in vitro studies. Incubation of primary rat hepatocytes with various carboxyl-containing compounds revealed no clear common factor which imparted potency as a peroxisomal inducer. Inhibitors of fatty acyl-CoA synthetase, norepinephrine and desulpho-CoA, however, decreased the level of peroxisomal induction by nafenopin in rat hepatocytes, suggesting that activation of carboxyl-containing compounds to their CoA thioesters may be a necessary step in initiating peroxisome proliferation. Coenzyme A thioesters of nafenopin, clofibric acid and other carboxyl-containing chemicals were synthesised and found to inhibit the activity of acetyl-CoA carboxylase to varying degrees. The CoA thioester of nafenopin was the most potent inhibitor among this group (Ki = 1.45 · 10−5 M), but weaker than palmitoyl-CoA (Ki = 2.22 · 10−6 M), the feedback inhibitor of acetyl-CoA carboxylase. Hypolipidaemia caused by treatment with peroxisome proliferators may, therefore, be related to inhibition of fatty-acid synthesis by the corresponding CoA thioester derivative.
Inhibition of palmitoyl co‐enzyme a hydrolase in mitochondria and microsomes by pharmaceutical organic anions
1990, Journal of Pharmaceutical SciencesRat microsomes and mitochondria were isolated and incubated with selected pharmaceutical organic anions at concentrations of 0, 0.2, 0.5, 1.0, and 2 mM. Activity of palmitoyl CoA hydrolase (PCAH) was shown to be reduced in a dose-dependent manner in microsomes by ibuprofen, valproate, acetyl salicylate, 2,4-dichlorophenoxyacetate (2,4-D), and 4-pentenoate, but not salicylate. Mitochondrial PCAH activity was inhibited by clofibrate, ibuprofen, valproate, and 2,4-D. Mitochondrial oxidative phosphorylation was impaired or uncoupled by each of the mitochondrial PCAH inhibitors. The inhibition of PCAH by some of these agents may lead to fatty acyl CoA accumulation. Very low concentrations of fatty acyl CoA are known to cause mitochondrial uncoupling and increase permeability. This action may play a role in the mitochondrial injury caused by some of these agents or related disease processes.
Purification and characterization of the enzymes of bile acid conjugation from fish liver
1990, Comparative Biochemistry and Physiology -- Part B: Biochemistry and- 1.
1. The two steps in bile acid conjugation have been studied in subcellular fractions of liver from three species of fish; vermillion rockfish, canary rockfish and ling codfish.
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2. The bile acid: coenzyme A (CoA) ligase activity in homogenates and isolated microsomes is undetectable due to indeterminate factors.
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3. A purification scheme is presented which eliminates the interfering factors. The purified ligase was found to have a lower affinity for bile acids as compared to the mammalian form and to be present in much lower titer.
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4. Since it appears to be the rate controlling enzyme in all species, it is expected that the rate of bile acid conjugation is much slower than in non-mammalian liver as compared to mammalian liver.
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5. The bile acid-CoA: taurin was found to exist as a dimer of molecular weight 100,000, in contrast to the monomeric mammalian forms.
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6. The only major kinetic difference is that the fish liver forms have rates of glycine conjugation which are only 1–2% of the rate with taurine, in part due to a very high for glycine.
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