Inhibition of ethanol-inducible cytochrome P450IIE1 by 3-amino-1,2,4-triazole

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Abstract

The p-nitrophenol hydroxylase activity of hepatic microsomes from acetone-treated rabbits was inhibited by 3-amino-1,2,4-triazole in a time- and NADPH-dependent manner. The loss of p-nitrophenol hydroxylation, an activity catalyzed predominately by P450IIE1, displayed a number of characteristics consistent with suicide inhibition of the enzyme. These include irreversibility, saturability, similarity of the effect of pH on the rate constant for inactivation and catalysis by the isozyme, protection by substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. At pH 6.8, the KI for 3-amino-1,2,4-triazole for inactivation was 57 mM and the maximal rate of inactivation was 0.43 min-1. The inactivation of hepatic microsomes resulted in a loss of spectrally detectable P450 which was correlated with the concentration of P450IIE1 in various microsomal preparations. Purified P450IIE1 was rapidly autoinactivated (kinact of about 0.1 min-1) in the presence of NADPH and P450 reductase. However, the autoinactivation was completely prevented by the addition of catalase. In the presence of catalase, purified P450IIE1 was inactivated in a time- and concentration-dependent manner by 3-amino-1,2,4-triazole (KI was 10 mM and the maximal rate of inactivation was 0.44 min-1). The inactivation resulted in the loss of spectrally detectable P450 but did not cause the formation of P420 or a loss of heme as determined by the reduced pyridine hemochrome. The spectrum of the inactivated enzyme exhibited a decreased extinction in the Soret region with a broad maximum at 378 nm and a shoulder around 428 nm. Inactivated P450IIE1 did not show a characteristic low-spin spectrum in the presence of 4-methylpyrazole. When 3-amino-1,2,4-[5-14C]triazole was used in the inactivation reaction, there was no significant incorporation of radioactivity into the protein or heme; these results suggest that the inactivation may be due to covalent binding of the heme to the protein or the modification of residues near the heme, which prevent substrate interaction. The effective inhibition of P450IIE1 by 3-amino-1,2,4-triazole suggests that the compound may be useful for the identification of P450IIE1-dependent microsomal catalysis.

Original languageEnglish (US)
Pages (from-to)377-383
Number of pages7
JournalChemical Research in Toxicology
Volume3
Issue number4
StatePublished - 1990
Externally publishedYes

Fingerprint

Amitrole
Cytochromes
Heme
Ethanol
Microsomes
Catalysis
Catalase
NADPH-Ferrihemoprotein Reductase
Hydroxylation
Nucleophiles
Triazoles
Liver
Radioactivity
Substrates
Enzymes
Acetone
Mixed Function Oxygenases
NADP
Suicide
Isoenzymes

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Chemistry(all)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

Inhibition of ethanol-inducible cytochrome P450IIE1 by 3-amino-1,2,4-triazole. / Koop, Dennis.

In: Chemical Research in Toxicology, Vol. 3, No. 4, 1990, p. 377-383.

Research output: Contribution to journalArticle

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abstract = "The p-nitrophenol hydroxylase activity of hepatic microsomes from acetone-treated rabbits was inhibited by 3-amino-1,2,4-triazole in a time- and NADPH-dependent manner. The loss of p-nitrophenol hydroxylation, an activity catalyzed predominately by P450IIE1, displayed a number of characteristics consistent with suicide inhibition of the enzyme. These include irreversibility, saturability, similarity of the effect of pH on the rate constant for inactivation and catalysis by the isozyme, protection by substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. At pH 6.8, the KI for 3-amino-1,2,4-triazole for inactivation was 57 mM and the maximal rate of inactivation was 0.43 min-1. The inactivation of hepatic microsomes resulted in a loss of spectrally detectable P450 which was correlated with the concentration of P450IIE1 in various microsomal preparations. Purified P450IIE1 was rapidly autoinactivated (kinact of about 0.1 min-1) in the presence of NADPH and P450 reductase. However, the autoinactivation was completely prevented by the addition of catalase. In the presence of catalase, purified P450IIE1 was inactivated in a time- and concentration-dependent manner by 3-amino-1,2,4-triazole (KI was 10 mM and the maximal rate of inactivation was 0.44 min-1). The inactivation resulted in the loss of spectrally detectable P450 but did not cause the formation of P420 or a loss of heme as determined by the reduced pyridine hemochrome. The spectrum of the inactivated enzyme exhibited a decreased extinction in the Soret region with a broad maximum at 378 nm and a shoulder around 428 nm. Inactivated P450IIE1 did not show a characteristic low-spin spectrum in the presence of 4-methylpyrazole. When 3-amino-1,2,4-[5-14C]triazole was used in the inactivation reaction, there was no significant incorporation of radioactivity into the protein or heme; these results suggest that the inactivation may be due to covalent binding of the heme to the protein or the modification of residues near the heme, which prevent substrate interaction. The effective inhibition of P450IIE1 by 3-amino-1,2,4-triazole suggests that the compound may be useful for the identification of P450IIE1-dependent microsomal catalysis.",
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N2 - The p-nitrophenol hydroxylase activity of hepatic microsomes from acetone-treated rabbits was inhibited by 3-amino-1,2,4-triazole in a time- and NADPH-dependent manner. The loss of p-nitrophenol hydroxylation, an activity catalyzed predominately by P450IIE1, displayed a number of characteristics consistent with suicide inhibition of the enzyme. These include irreversibility, saturability, similarity of the effect of pH on the rate constant for inactivation and catalysis by the isozyme, protection by substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. At pH 6.8, the KI for 3-amino-1,2,4-triazole for inactivation was 57 mM and the maximal rate of inactivation was 0.43 min-1. The inactivation of hepatic microsomes resulted in a loss of spectrally detectable P450 which was correlated with the concentration of P450IIE1 in various microsomal preparations. Purified P450IIE1 was rapidly autoinactivated (kinact of about 0.1 min-1) in the presence of NADPH and P450 reductase. However, the autoinactivation was completely prevented by the addition of catalase. In the presence of catalase, purified P450IIE1 was inactivated in a time- and concentration-dependent manner by 3-amino-1,2,4-triazole (KI was 10 mM and the maximal rate of inactivation was 0.44 min-1). The inactivation resulted in the loss of spectrally detectable P450 but did not cause the formation of P420 or a loss of heme as determined by the reduced pyridine hemochrome. The spectrum of the inactivated enzyme exhibited a decreased extinction in the Soret region with a broad maximum at 378 nm and a shoulder around 428 nm. Inactivated P450IIE1 did not show a characteristic low-spin spectrum in the presence of 4-methylpyrazole. When 3-amino-1,2,4-[5-14C]triazole was used in the inactivation reaction, there was no significant incorporation of radioactivity into the protein or heme; these results suggest that the inactivation may be due to covalent binding of the heme to the protein or the modification of residues near the heme, which prevent substrate interaction. The effective inhibition of P450IIE1 by 3-amino-1,2,4-triazole suggests that the compound may be useful for the identification of P450IIE1-dependent microsomal catalysis.

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