The spontaneous and enzymatic reaction of N-acetyl-p-benzoquinonimine with glutathione: A stopped-flow kinetic study

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Abstract

The spontaneous and glutathione (GSH) transferase catalyzed reactions of GSH and N-acetyl-p-benzoquinonimine (NABQI) have been studied by stopped-flow kinetics. The spontaneous reaction was shown to be first order in NABQI, GSH and inversely proportional to the H+ concentration; e.g., at pH 7.0 and 25 °C the second-order rate constant was 3.2 × 104m−1s−1. Data for the enzymatic reaction gave values for Km of 27, 1.3, 7, and 7 μm and values for kcat of 90,37,5.1, and 165 s−1 for rat liver GSH transferases 1-1, 2-2, 3-3, and 7-7, respectively. Over a wide range of reactant concentrations and pH, the spontaneous reaction yields three products, namely a GSH conjugate, 3-(glutathion-S-yl)acetaminophen; a reduction product, acetaminophen; and an oxidation product, glutathione disulfide in the proportions 2:1:1. Analysis of products formed after enzymatic reaction showed that both GSH conjugation and the reduction of NABQI to acetaminophen were catalyzed to an extent characteristic of each isoenzyme. With respect to GSH conjugation, GSH transferase isoenzymes were effective in the order 7-7 > 2-2 > 1-1 > 3-3 > 4-4, and with respect to NABQI reduction these isoenzymes were effective in the order 1-1 > 2-2 > 7-7 the position of isoenzymes 3-3 and 4-4 being uncertain. Human GSH transferases δ, μ, and π behave similarly to the homologous rat enzymes, i.e., toward conjugation in the order π > δ > μ and reduction δ > μ > π (for nomenclature see W. B. Jakoby, B. Ketterer, and B. Mannervik, (1984) Biochem. Pharmacol.33, 2539–2540). Possible mechanisms of the reaction and its effect on the toxicity of NABQI are discussed.

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