Mixed function oxidases from germinating castor bean endosperm

Homogenates from germinating castor bean endosperm were fractionated by sucrose density gradient centrifugation and examined for mixed function oxidase activity. Activity of cinnamic acid 4-hydroxylase and p-chloro-N-methylaniline N-demethylase was highest in the endoplasmic reticulum fraction. Activity of both enzymes is dependent on NADPH and on molecular oxygen; both activities are inhibited by carbon monoxide. When challenged with a number of potential inhibitors the enzymes responded in ways fairly typical of mixed function oxidases from other plants and animals. The N-demethylase appears to be specific for N-methylarylamines. In the absence of NADPH, cumene hydroperoxide is able to support N-demethylation. The mechanistic significance of this activity is discussed.     

Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase

N-demethylases have been reported to remove the methyl groups on primary or secondary amines, which could further affect the properties and functions of biomacromolecules or chemical compounds; however, the substrate scope and the robustness of N-demethylases have not been systematically investigated. Here we report the recreation of natural evolution in key microdomains of the Thermomicrobium roseum sarcosine oxidase (TrSOX), an N-demethylase with marked stability (melting temperature over 100 °C) and enantioselectivity, for enhanced substrate scope and catalytic efficiency on -C-N- bonds. We obtained the structure of TrSOX by crystallization and X-ray diffraction (XRD) for the initial framework. The natural evolution in the nonconserved residues of key microdomains—including the catalytic loop, coenzyme pocket, substrate pocket, and entrance site—was then identified using ancestral sequence reconstruction (ASR), and the substitutions that accrued during natural evolution were recreated by site-directed mutagenesis. The single and double substitution variants catalyzed the N-demethylation of N-methyl-L-amino acids up to 1800- and 6000-fold faster than the wild type, respectively. Additionally, these single substitution variants catalyzed the terminal N-demethylation of non-amino-acid compounds and the oxidation of the main chain -C-N- bond to a -C=N- bond in the nitrogen-containing heterocycle. Notably, these variants retained the enantioselectivity and stability of the initial framework. We conclude that the variants of TrSOX are of great potential use in N-methyl enantiomer resolution, main-chain Schiff base synthesis, and alkaloid modification or degradation.     

Caffeine Junkie: an Unprecedented Glutathione S-Transferase-Dependent Oxygenase Required for Caffeine Degradation by Pseudomonas putida CBB5

Caffeine and other N-methylated xanthines are natural products found in many foods, beverages, and pharmaceuticals. Therefore, it is not surprising that bacteria have evolved to live on caffeine as a sole carbon and nitrogen source. The caffeine degradation pathway of Pseudomonas putida CBB5 utilizes an unprecedented glutathione-S-transferase-dependent Rieske oxygenase for demethylation of 7-methylxanthine to xanthine, the final step in caffeine N-demethylation. The gene coding this function is unusual, in that the iron-sulfur and non-heme iron domains that compose the normally functional Rieske oxygenase (RO) are encoded by separate proteins. The non-heme iron domain is located in the monooxygenase, ndmC, while the Rieske [2Fe-2S] domain is fused to the RO reductase gene, ndmD. This fusion, however, does not interfere with the interaction of the reductase with N₁- and N₃-demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N₇-demethylation reaction absolutely requires a unique, tightly bound protein complex composed of NdmC, NdmD, and NdmE, a novel glutathione-S-transferase (GST). NdmE is proposed to function as a noncatalytic subunit that serves a structural role in the complexation of the oxygenase (NdmC) and Rieske domains (NdmD). Genome analyses found this gene organization of a split RO and GST gene cluster to occur more broadly, implying a larger function for RO-GST protein partners.     

The Novel Bacterial N-Demethylase PdmAB Is Responsible for the Initial Step of N,N-Dimethyl-Substituted Phenylurea Herbicide Degradation

The environmental fate of phenylurea herbicides has received considerable attention in recent decades. The microbial metabolism of N,N-dimethyl-substituted phenylurea herbicides can generally be initiated by mono-N-demethylation. In this study, the molecular basis for this process was revealed. The pdmAB genes in Sphingobium sp. strain YBL2 were shown to be responsible for the initial mono-N-demethylation of commonly used N,N-dimethyl-substituted phenylurea herbicides. PdmAB is the oxygenase component of a bacterial Rieske non-heme iron oxygenase (RO) system. The genes pdmAB, encoding the α subunit PdmA and the β subunit PdmB, are organized in a transposable element flanked by two direct repeats of an insertion element resembling ISRh1. Furthermore, this transposable element is highly conserved among phenylurea herbicide-degrading sphingomonads originating from different areas of the world. However, there was no evidence of a gene for an electron carrier (a ferredoxin or a reductase) located in the immediate vicinity of pdmAB. Without its cognate electron transport components, expression of PdmAB in Escherichia coli, Pseudomonas putida, and other sphingomonads resulted in a functional enzyme. Moreover, coexpression of a putative [3Fe-4S]-type ferredoxin from Sphingomonas sp. strain RW1 greatly enhanced the catalytic activity of PdmAB in E. coli. These data suggested that PdmAB has a low specificity for electron transport components and that its optimal ferredoxin may be the [3Fe-4S] type. PdmA exhibited low homology to the α subunits of previously characterized ROs (less than 37% identity) and did not cluster with the RO group involved in O- or N-demethylation reactions, indicating that PdmAB is a distinct bacterial RO N-demethylase.     

Induction and suppression of hepatic and extrahepatic microsomal foreign-compound-metabolizing enzyme systems by 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on a number of hepatic and extrahepatic foreign-compound-metabolizing enzyme systems in microsomes from rats, rabbits and guinea pigs were investigated.Following TCDD treatment, the N-demethylation of benzphetamine, aminopyrine and ethylmorphine was suppressed in hepatic microsomes from male but not from female rats. However, both cytochrome P-450 and benzpyrene hydroxylase were significantly stimulated in hepatic microsomes from both male and female rate at doses as small as 1 μg TCDD/kg body weight. The inductive effect on rat hepatic microsomal enzymes was considerably more persistent than the suppressive effect. Following a single oral dose of 25 μg TCDD/kg body weight, benzpyrene hydroxylase of male rat liver microsomes remained significantly elevated for 73 days but the suppression of benzphetamine N-demethylase had gone after 35 days.The induction of benzpyrene hydroxylase in male rat liver microsomes by TCDD was independent of the age of the rat and the levels to which this enzyme was increased was similar in male rats of all ages. However, the suppression of benzphetamine N-demethylase in male rat liver microsomes was age related: the suppression was seen only in adult animals and in the very young (10 days old) the enzyme was actually induced by TCDD. Inductive effects appeared in both smooth and rough-surfaced hepatic microsomes from male rats but the suppression of N-demethylidon occurred perhaps the derepression arises through the interaction of TCDD or metabolite of TCDD, with the operator gene itself.     

Cytochromes P450 from Papilio polyxenes: Adaptations to host plant allelochemicals

1. Papilio polyxenes, a caterpillar which feeds on xanthotoxin-containing plants, has cytochromes P450 that are six- to 100-fold less sensitive to the suicide substrate inhibitor, xanthotoxin, than cytochromes P450 from Manduca sexta, which does not survive on xanthotoxin-containing plants.2. Xanthotoxin is a suicide substrate inhibitor of O-demethylation of p-nitroanisole by M. sexta microsomes but a reversible inhibitor of O-demethylation by P. polyxenes microsomes.3. Aldrin epoxidation is irreversibly inhibited by xanthotoxin in both species.4. Patterns of cross inhibition demonstrate that O-demethylase and aldrin epoxidase from both species and the P. polyxenes xanthotoxin-metabolizing cytochrome P450 are distinct enzymes.     

Novel Three-Component Rieske Non-Heme Iron Oxygenase System Catalyzing the N-Dealkylation of Chloroacetanilide Herbicides in Sphingomonads DC-6 and DC-2

Sphingomonads DC-6 and DC-2 degrade the chloroacetanilide herbicides alachlor, acetochlor, and butachlor via N-dealkylation. In this study, we report a three-component Rieske non-heme iron oxygenase (RHO) system catalyzing the N-dealkylation of these herbicides. The oxygenase component gene cndA is located in a transposable element that is highly conserved in the two strains. CndA shares 24 to 42% amino acid sequence identities with the oxygenase components of some RHOs that catalyze N- or O-demethylation. Two putative [2Fe-2S] ferredoxin genes and one glutathione reductase (GR)-type reductase gene were retrieved from the genome of each strain. These genes were not located in the immediate vicinity of cndA. The four ferredoxins share 64 to 72% amino acid sequence identities to the ferredoxin component of dicamba O-demethylase (DMO), and the two reductases share 62 to 65% amino acid sequence identities to the reductase component of DMO. cndA, the four ferredoxin genes, and the two reductases genes were expressed in Escherichia coli, and the recombinant proteins were purified using Ni-affinity chromatography. The individual components or the components in pairs displayed no activity; the enzyme mixture showed N-dealkylase activities toward alachlor, acetochlor, and butachlor only when CndA-His₆ was combined with one of the four ferredoxins and one of the two reductases, suggesting that the enzyme consists of three components, a homo-oligomer oxygenase, a [2Fe-2S] ferredoxin, and a GR-type reductase, and CndA has a low specificity for the electron transport component (ETC). The N-dealkylase utilizes NADH, but not NADPH, as the electron donor.     

Electron paramagnetic resonance study of the oxidation of N-methyl-substituted aromatic amines catalyzed by hemeproteins

The oxidation of N-mono- and dimethyl-substituted toluidines and aniline by H₂O₂, catalyzed by horseradish peroxidase or metmyoglobin, produces organic free radicals, detectable by electron paramagnetic resonance spectroscopy at room temperature. The radical cation of N,N-dimethyl-p-toluidine was conclusively identified, but the other resolved EPR signals were assigned to radical cations of radical dimerization products, e.g., N,N,N′,N′-tetramethylbenzidine formed from N,N-dimethylaniline. The N-demethylase activities of metmyoglobin were found to be uniformly smaller than those of horseradish peroxidase, consistent with the much faster reaction of the latter hemeprotein with H₂O₂. Detection of the monomeric radical cation of N,N-demethyl-p-toluidine correlated with the largest rate of N-demethylation among this class of compounds. These findings emphasize the importance of radical stability (provided, for example, by the para methyl substituent) on subsequent competing reactions of the radical cation of the N-methyl substrate, i.e., one-electron oxidation leading to formaldehyde release or radical dimerization, which becomes more probable for the less stable radical intermediates. Attempts were made to correlate these results with data obtained for the $\text{O}{}_2\text{NADPH}\text{-supported}$ oxidation of these same substrates by liver microsomal cytochrome P-450. However, pronounced differences in physical state and kinetic properties of this heterogeneous, membrane-associated microsomal hemeprotein and the soluble “model” hemeprotein systems precluded firm conclusions concerning a radical mechanism of N-demethylation monooxygenase activities of microsomal fractions.     

Effect of succinate on aminopyrine N-demethylase activity

Succinate stimulated the aminopyrine N-demethylase activity in the presence of rate-limiting concentrations of NADPH in the crude mitochondrial preparation, as well as in a reconstituted system containing microsomes and microsome-free mitochondria. The increase in enzyme activity was accompanied by a decrease of the apparent K_m of NADPH.The stimulating effect of succinate was counteracted by malonate, rotenone and pentachlorophenol. No significant formaldehyde oxidation was exhibited by the crude mitochondrial preparation under the conditions of N-demethylase assay.Enhancement of the N-demethylase activity by succinate is supposed to be due to a mitochondrial-microsomal interaction which may play a role in the regulation of drug metabolism.     

Genetic expression of aryl hydrocarbon hydroxylase induction. VI. Control of other aromatic hydrocarbon-inducible mono-oxygenase activities at or near the same genetic locus

When mice are administered aromatic hydrocarbons, the induction of aryl hydrocarbon (benzo[a]pyrene) hydroxylase, p-nitroanisole O-demethylase, 7-ethoxycoumarin O-deethylase, and 3-methyl-4-methylaminoazobenzene N-demethylase activities—all membrane-bound mono-oxygenases having cytochrome P₄₅₀ associated with their active sites—is associated with the same genetic locus or with closely linked loci; we have previously proposed that this genetic region be designated the Ah locus for aromatic hydrocarbon responsiveness. Expression of these four inducible enzyme activities occurs as a single autosomal dominant trait in offspring from a genetic cross between inbred C57BL/6N and DBA/2N mice and from the appropriate backcrosses and intercross. There are no striking differences in relative thermolability or ontogenetic expression among these four closely linked aromatic hydrocarbon-induced mono-oxygenase activities. All four of these microsomal enzyme activities exist in two forms—one predominantly present in control or aromatic hydrocarbon-treated genetically nonresponsive mice and the other predominantly present in aromatic hydrocarbon-treated genetically responsive mice; the latter form is preferentially inhibited in vitro by such compounds as α-naphthoflavone. Whether a single induction-specific protein or a group of induction-specific proteins is associated with the Ah locus remains uncertain. The expression of aminopyrine N-demethylase, d-benzphetamine N-demethylase, NADPH-cytochrome c reductase, and NADPH-cytochrome P₄₅₀ reductase activities in aromatic hydrocarbon-treated genetically responsive and nonresponsive mice is not correlated with the Ah locus.     

Evidence for a free radical mechanism of N-demethylation of N,N-dimethylaniline and an analog by hemeprotein--H2O2 systems.

Horseradish peroxidase and metmyoglobin catalyze the H₂O₂-supported N-demethylation of N,N-dimethylaniline and N,N-dimethyl-p-toluidine. The catalytic activities of horseradish peroxidase are more than 100-fold larger than those of metmyoglobin or those previously reported for liver microsomal cytochrome P-450. Distinct free radical species of these N-methyl substrates were detected with both catalysts. These findings establish the general validity of a recently proposed free radical mechanism of oxidative N-demethylation (Griffin, B. W., and Ting, P. L., Biochemistry (1978), 2206–2211), which is quite different from that previously suggested for the analogous cytochrome P-450-dependent reactions.     

Hydroperoxide specificity of plant and human tissue lipoxygenase: an in vitro evaluation using N-demethylation of phenothiazines

Since hydroperoxide specificity of lipoxygenase (LO) is poorly understood at present, we investigated the ability of cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) to support cooxidase activity of the enzyme toward the selected xenobiotics. Considering the fact that in the past, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases, in this study, we investigated the ability of non-heme iron proteins, namely soybean LO (SLO) and human term placental LO (HTPLO) to mediate N-demethylation of phenothiazines. In addition to being dependent on peroxide concentration, the reaction was dependent on enzyme concentration, substrate concentration, incubation time, and pH of the medium. Using Nash reagent to estimate formaldehyde production, the specific activity under optimal assay conditions for the SLO mediated N-demethylation of chlorpromazine (CPZ), a prototypic phenothiazine, in the presence of TBHP, was determined to be 117±12 nmol HCHO/min/mg protein, while that of HTPLO was 3.9±0.40 nmol HCHO/min/mg protein. Similar experiments in the presence of CHP yielded specific activities of 106±11 nmol HCHO/min/mg SLO, and 3.2±0.35 nmol HCHO/min/mg HTPLO. As expected, nordihydroguaiaretic acid and gossypol, the classical inhibitors of LOs, as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of formaldehyde production from CPZ by SLO in the reaction media fortified with either CHP or TBHP. Besides chlorpromazine, both SLO and HTPLO also mediated the N-demethylation of other phenothiazines in the presence of these organic hydroperoxides.     

A comparative study of liver mixed function oxidases in camels (Camelus dromedarius), guinea pigs (Cavia porcellus) and rats (Rattus norvegicus)

1. The activities of the drug-metabolizing enzymes, benzphetamine N-demethylase, 7-ethoxy-coumarin O-deethylase and dicoumarol oxidation have been measured in vitro in the liver of camels, guinea pigs and rats.2. In these species, levels of hepatic microsomal parameters namely microsomal protein, cytochrome P₄₅₀, cytochrome b₅ and NADPH-cytochrome c reductase have also been determined.3. In general, camels seemed to have the lowest enzyme activity when compared to rats and guinea pigs.4. Some sex differences were observed in the levels of enzymes studied. In rats and guinea pigs, males had higher benzphetamine N-demethylase than females. However, in camels and guinea pigs, females had higher 7-ethoxycoumarin O-deethylase when compared to males.     

Effects of methylbenzenes on microsomal enzymes in rat liver,kidney and lung

The effects of pretreatment with toluene, o-, m-, p-xylene and mesitylene were investigated on the microsomal enzymes of liver, kidney and lung in rats. The activities of aminopyrine N-demethylase, aryl hydrocarbon hydroxylase, aniline hydroxylase, NADPH-cytochrome c reductase, as well as the concentrations of cytochrome P-450 and cytochrome b₅ were determined. The effects were most marked in the liver, where toluene caused increase in aniline hydroxylase and cytochrome P-450; o-xylene in aminopyrine N-demethylase and cytochrome b₅; m-xylene and mesitylene in all the enzymes investigated. In kidneys, all the compounds increased the activity of aniline hydroxylase; m-xylene induced cytochrome P-450 and b₅ as well as NADPH-cytochrome c reductase; p-xylene induced cytochrome P-450, and mesitylene cytochrome P-450 and b₅. Aminopyrine N-demethylase activity was decreased by toluene. In lungs, only mesitylene caused any significant differences from the controls: increase in aminopyrine N-demethylase and aryl hydrocarbon hydroxylase, decrease in aniline hydroxylase. The methylbenzenes tested induced the microsomal enzymes in a rough correlation to the number of their methyl groups and their hydrophobic properties.     

Radical mechanism of aminopyrine oxidation by cumene hydroperoxide catalyzed by purified liver microsomal cytochrome P-450

Under identical experimental conditions, purified preparations of rabbit liver microsomal cytochrome P-450 and beef heart metmyoglobin were equally effective at stimulating the oxidation of aminopyrine to a free radical species by cumene hydroperoxide. Mannitol had no effect on radical levels produced with either hemeprotein-hydroperoxide system; however, specific ligands of the two hemeproteins, substrates of cytochrome P-450, and phospholipid affected the two systems quite differently. Only the metmyo-globindependent oxidation of aminopyrine was significantly inhibited by fluoride and cyanide. Metyrapone, a specific ligand of cytochrome P-450, and benzphetamine, which was N-demethylated by cumene hydroperoxide only in the presence of cytochrome P-450, inhibited only the cytochrome P-450-stimulated oxidation of aminopyrine. Moreover, only with the solubilized liver hemeprotein was aminopyrine radical generation markedly stimulated by phospholipid. Similar properties of aminopyrine N-demethylation and radical formation by the cytochrome P-450-cumene hydroperoxide system have strongly implicated the radical as a requisite intermediate in product formation. Micromolar concentrations of metyrapone caused parallel inhibition, by at least 50%, of both radical generation and formaldehyde production. These results support a radical pathway of N-demethylation proposed for other hemeprotein-hydroperoxide systems (B. W. Griffin and P. L. Ting, 1978, Biochemistry, 17, 2206–2211), in which the substrate undergoes two successive one-electron abstractions, followed by hydrolysis of the iminium cation intermediate. Thus, for this class of substrates, the experimental data are consistent with the oxygen atom of the product arising from H₂O and not directly from the hydroperoxide, which has been previously proposed as a general mechanism for cytochrome P-450 peroxidatic activities.     

Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex

Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as Pseudomonas putida CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial N-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial N₁- and N₃-specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper N-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during N-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications.     

Protein Hydroxylation Catalyzed by 2-Oxoglutarate-dependent Oxygenases

The post-translational hydroxylation of prolyl and lysyl residues, as catalyzed by 2-oxoglutarate (2OG)-dependent oxygenases, was first identified in collagen biosynthesis. 2OG oxygenases also catalyze prolyl and asparaginyl hydroxylation of the hypoxia-inducible factors that play important roles in the adaptive response to hypoxia. Subsequently, they have been shown to catalyze N-demethylation (via hydroxylation) of N^ϵ-methylated histone lysyl residues, as well as hydroxylation of multiple other residues. Recent work has identified roles for 2OG oxygenases in the modification of translation-associated proteins, which in some cases appears to be conserved from microorganisms through to humans. Here we give an overview of protein hydroxylation catalyzed by 2OG oxygenases, focusing on recent discoveries.     

A sensitive,fluorometric method for the assay of microsomal hydroxylase: N-demethylation of p-chloro-N-methylaniline

A fluorometric method for the assay of microsomal hydroxylase activity is described. N-Demethylation of p-chloro-N-methylaniline yields p-chloroaniline, which is coupled with fluorescamine, extracted with ethylacetate, and measured fluorometrically. This method can determine low levels of N-demethylase activity.     

Author index to volume 154

Horseradish peroxidase-catalyzed N-demethylation of aminopyrine and dimethylaniline results in generation of free radical intermediates which can interact with glutathione (GSH) to form a glutathione radical. This can either dimerize to yield glutathione disulfide or react with O₂ to form oxygenated products of glutathione. Ethylmorphine is not a substrate in the peroxidase-mediated reaction, and free radical intermediates which react with GSH, are not formed from aminopyrine and dimethylaniline when the horseradish peroxidase/H₂O₂ system is replaced by liver microsomes and NADPH. Therefore, it appears unlikely that formation of free radical intermediates can be responsible for the depletion of GSH observed during N-demethylation of several drugs in isolated liver cells.     

Liver metabolic reactions: tertiary amine N-dealkylation, tertiary amine N-oxidation, N-oxide reduction, and N-oxide N-dealkylation. II. N,N-dimethylaniline

Rat liver microsomal preparations enzymatically catalyze the N-demethylation and N-oxidation of dimethylaniline as well as the N-demethylation of dimethylaniline-N-oxide. Both compounds were used as substrates and the formation of formaldehyde and N-oxide were determined.Both demethylation and N-oxidation of dimethylaniline are dependent on NADPH. This cofactor also increases the demethylation of dimethylaniline-N-oxide, although it is not an absolute requirement. Nicotinamide increases the rate of formation of formaldehyde and N-oxide from dimethylaniline by a factor of about 4 and decreases the N-oxide demethylation by the same factor. The cofactor optimum consists of NADPH, nicotinamide, and magnesium ions for the demethylation and N-oxidation of dimethylaniline, and of NADPH alone for the demethylation of its N-oxide. The kinetic constants of the three test reactions have been determined under these optimal cofactor requirements.Various agents strongly influence the rates of product formation of the three test reactions studied. SH-blocking agents, the chelating agent EGTA, as well as nicotinamide influence the rates of formaldehyde formation from dimethylaniline and N-oxide demethylation in an opposite way. This demonstrates that, in the tertiary amine demethylation of dimethylaniline, a C-oxidation pathway is operative in addition to an N-oxidation pathway with subsequent N-oxide demethylation. The following influences on the actual metabolic reactions could be deduced from the effects of agents on the test reactions: SKF 525-A inhibits and phenobarbital pretreatment stimulates N-oxide demethylation; EDTA inhibits both the latter reaction and N-oxidation; EGTA and nicotinamide stimulate C-oxidation and inhibit N-oxide demethylation; SH-blocking agents inhibit C-oxidation and stimulate both N-oxidation and N-oxide demethylation.Quantitative and qualitative species differences with respect to cofactor requirement and effect of SKF 525-A have been observed between rat and pig liver microsomes. In addition, profound differences in subcellular localization and metabolic rates between dimethylaniline and other substrates are known. Thus it is unlikely that the three metabolic reactions dealt with in this report are characteristic of tertiarr amine N-dealkylation in general.