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PubMed1984

Inhibition of rat hepatic mixed function oxidases by antimalarial drugs: selectivity for cytochromes P-450 and P-448.

Intraperitoneal administration of chloroquine, primaquine and quinacrine to rats resulted in inhibition of the hepatic microsomal mixed-function oxidases. The N-demethylation of benzphetamine (cytochrome P-450) was inhibited by chloroquine only while the O-deethylation of ethoxyresorufin (cytochrome P-448) was inhibited by primaquine and quinacrine. When incubated with hepatic microsomes from phenobarbital-pretreated rats, chloroquine and primaquine, but not quinacrine, caused a concentration-dependent inhibition of benzphetamine N-demethylase activity. Incubation of hepatic microsomes from beta-naphthoflavone rats with primaquine and quinacrine, but not chloroquine, resulted in a concentration-dependent inhibition of the O-deethylation of ethoxyresorufin. These observations demonstrate that chloroquine and quinacrine are specific inhibitors of cytochromes P-450 and P-448, respectively.

Animals↗
PubMed1990

Electrophoretic, spectral, catalytic and immunochemical properties of highly purified cytochrome P-450 from sheep lung.

1. Cytochrome P-450LgM2 was purified from sheep lung microsomes in the presence of detergents, Emulgen 913 and cholate. 2. The purification procedure involved the chromatography of the detergent solubilized microsomes on DEAE-cellulose and hydroxylapatite. 3. Cytochrome P-450LgM2 was further purified on second DEAE-cellulose and hydroxylapatite columns. 4. The specific content of the highly purified P-450LgM2 was 16-18 nmol P-450/mg protein and purified 164-fold. 5. The yield was 16% of the initial content in microsomes. 6. The SDS-polyacrylamide slab gel electrophoresis (PAGE) of the purified lung cytochrome P-450LgM2 showed one protein band having the monomer molecular weight of 49,500. 7. The absolute CO-difference spectrum of dithionate-reduced P-450LgM2 gave a peak at 451 nm. 8. When sheep lung cytochrome P-450LgM2 and P-450LM2 purified from liver of phenobarbital (PB)-induced rabbit were subjected to Western Blotting and visualized immunochemically with anti-P-450LM2, they showed identical mobilities. 9. P-450LgM2 was found to be very active in N-demethylation of benzphetamine in a reconstituted system containing purified sheep lung reductase and synthetic lipid. 10. Turnover numbers (min-1) for benzphetamine, aniline, ethylmorphine and p-nitrophenol were determined to be 273, 1.2, 15.5 and 1.05, respectively, in a reconstituted microsomal lung monooxygenase system. 11. Spectral, electrophoretic, biocatalytic and immunochemical properties of sheep lung P-450LgM2 were found to be similar to those of P-450 isozyme 2, purified from PB-treated rabbit liver and of rabbit lung microsomes.

Animals↗
PubMed1995

Comparison of hepatic and extrahepatic drug-metabolizing enzyme activities in rats given single or multiple challenge infections with Fasciola hepatica.

The activity of drug-metabolizing enzymes was compared in liver, kidneys and lungs of rats given single or repetitive fluke infections. Fascioliasis was induced by oral administration of 20 metacercariae of F. hepatica to rats, either 6, or 12 and 6, or 12, 9 and 6 weeks before sacrifice. In the liver of mono-infected rats, significant reductions (24-67%) were observed in microsomal content of cytochrome P450 and all P450-dependent monooxygenases investigated. Conjugations to glutathione or acetate were lowered by 34-50% in these animals. In multiply infected animals, a majority of specific enzymatic activities were unchanged, while some monooxygenase activities such as aminopyrine demethylation or benzo(a)pyrene hydroxylation were increased by 26-76% in the liver of tri-infected rats. A renal compensatory process occurred in all infected groups, since cytochrome P450, benzphetamine demethylation and glutathione conjugation were significantly increased. By contrast, dealkylation of benzphetamine and pentoxyresorufin were decreased in the lungs of monoinfected rats. The development of parasite resistance would account for the recovery of liver drug metabolizing capabilities in multi-infected animals.

Animals↗
PubMed1988

The effect of long-term streptozotocin-induced diabetes on the hepatotoxicity of bromobenzene and carbon tetrachloride and hepatic biotransformation in rats.

To exclude the possibility that changes in hepatotoxicity and biotransformation were induced by diabetogen administration, the influence of long-lasting experimental insulin-dependent diabetes on the activities of benzphetamine demethylase, styrene oxide hydrolase, and UDP-glucuronosyl-transferases toward 1-naphthol, diethylstilbestrol, estrone and testosterone, and glutathione S-transferases toward 1-chloro-2,4-dinitrobenzene, ethacrynic acid, and sulfobromophthalein was studied. Adult male Sprague-Dawley rats injected with 45 mg streptozotocin/kg rapidly developed the classical symptoms of diabetes which persisted throughout the 90-day test period. Ketonemia was detectable at 6 but not at either 35 or 90 days after streptozotocin administration. After acute challenge with bromobenzene or carbon tetrachloride (CCl4), aspartate and alanine aminotransferase activities in rats diabetic for 35 and 90 days were markedly higher than those in normal rats, suggesting that diabetes potentiated the hepatotoxicity of these chemicals. Administration of 25 microliters CCl4/kg, ip, to diabetic rats decreased enzyme activities toward benzphetamine, sulfobromophthalein, 1-chloro-2,4-dinitrobenzene, and 1-naphthol. In normal rats, a dose of 400 microliters CCl4/kg, ip, was required to cause similar changes in enzyme activities. Bromobenzene (500 microliters/kg, ip) elicited opposing responses in diabetic and normal rats in N-demethylase activity, in UDP-glucuronosyltransferase activity toward 1-naphthol, estrone, and testosterone, and in glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene. Total cytochrome P450 concentrations were reduced by both induction of diabetes and hepatotoxicant challenge. Thus, chronic uncontrolled diabetes alters the response of hepatic xenobiotic biotransformation enzymes in a non-uniform, substrate-dependent manner, independent of initial diabetogen effects. The role of cytochrome P450j in potentiating CCl4 toxicity is discussed.

Animals↗
PubMed1989

Effects of ethanol on microsomal drug metabolism in aging female rats. I. Induction.

The purpose of this study was to determine how aging affects the induction by ethanol or acetone of the hepatic microsomal monooxygenase system of female Fischer 344 rats. Young-adult, middle-aged and old rats (4, 14 and 25 months) were fed an ethanol-containing or control liquid diet for 15 days. Cytochrome P-450, cytochrome c reductase, aniline hydroxylase, nitrophenol hydroxylase, nitroanisole O-demethylase and benzphetamine N-demethylase activities were measured in hepatic microsomes. All of the drug metabolism activities except benzphetamine N-demethylase were 20-35% lower in old than in young-adult rats fed the control diet. In addition, the increase in drug metabolism produced by feeding the regular ethanol diet (36% of calories as ethanol) was 50-60% lower in the old rats. However, there was no difference in the magnitude of ethanol induction when ethanol intakes were matched. The effects of chronic acetone consumption (1.2g/day per kg body weight for 15 days) paralleled those of ethanol consumption, except that the extent of induction was greater with acetone. Acetone-induced levels of hepatic microsomal cytochrome P-450, nitrophenol hydroxylase, nitroanisole O-demethylase and aniline hydroxylase were similar in all three age groups. The results of this study indicate that induction of hepatic microsomal drug metabolism by ethanol or acetone is unaffected by the aging process.

Acetone↗
PubMed1983

Modification of cytochrome P-450 with fluorescein isothiocyanate.

Fluorescein isothiocyanate (FITC) has been shown to be selectively attached to the N-terminus of cytochrome P-450 LM2. The N-demethylase activity of cytochrome P-450 LM2 reconstituted systems modified in this way was inhibited by 25%. As revealed by CD measurements the overall conformation as well as the immediate heme environment of cytochrome P-450 LM2 remained unchanged after attachment of the FITC molecule. The binding affinity of modified cytochrome P-450 LM2 toward benzphetamine and aniline and the cumene hydroperoxide- or H2O2-supported N-demethylation of benzphetamine are maintained. However, the introduction of the electron via NADPH-cytochrome P-450 reductase (EC 1.6.2.4) is impaired after modification of the alpha-amino group. The extent of reduced modified cytochrome P-450 LM2 in the cytochrome P-450 reductase-supported reduction reaction is diminished and the half-time of the reduction is increased. The diminished reducibility is ascribed to steric hindrance of groups directly involved in the interaction between cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase or to blocking of the charge-pair interactions between the alpha-amino group of P-450 LM2 and the respective negatively charged group of NADPH-cytochrome P-450 reductase. By energy-transfer measurements distances between the heme and the alpha-amino group of 2.65 and 3.97 nm for the oligomeric and the monomeric forms of P-450 LM2, respectively, have been determined.

Animals↗
PubMed1990

Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450tu) from mouse liver tumors.

Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.

Amino Acid Sequence↗
PubMed1985

Foetal and neonatal development of cytochrome P-450 and cytochrome P-448 catalysed mixed function oxidases in the rat: induction by 3-methylcholanthrene.

Benzphetamine N-demethylase (cytochrome P-450) and ethoxyresorufin O-deethylase activities (cytochrome P-448) were determined in the growing neonate and foetus of control and 3-methylcholanthrene-pretreated rats. Ethoxyresorufin O-deethylase activity was highest in the 1-2-week-old animals and then decreased with age. The inducibility of this activity by 3-methylcholanthrene was low in the young animals, but increased with age. In contrast, benzphetamine N-demethylase activity in the control animals was low at birth and increased with age, and was not induced by 3-methylcholanthrene. In the foetal liver, ethoxyresorufin O-deethylase was the only activity present at higher levels than in the maternal liver. Transplacental administration of 3-methylcholanthrene failed to induce the foetal activities while the maternal liver showed the expected response. These observations demonstrate that cytochrome P-448 may be a predominant hepatic form in the foetus and neonate but cytochrome P-450 becomes a major form as the animal grows. The implications of these findings in chemical toxicity are discussed.

Age Factors↗
PubMed1992

Alterations in hepatic phase I and phase II biotransformation enzymes by garlic oil in rats.

Studies were conducted to examine the effect of a single and repeated administrations of garlic oil (diallyl sulfide) on Phase I and Phase II biotransformation enzymes in rats. Adult, male Sprague-Dawley rats treated with a single dose of garlic oil (500 mg/kg i.p.) showed a significant depression of hepatic cytochrome P-450, aminopyrine N-demethylase and aniline hydroxylase while microsomal protein content, cytochrome b5, NADPH-cytochrome c reductase, benzphetamine N-demethylase and cytosolic glutathione, S-transferase remained unaffected 24 h following the treatment. Although certain microsomal enzymes were depressed, there was no liver damage caused by garlic oil as judged by the putative serum enzyme test. On the other hand, daily administration of garlic oil (50 mg/kg i.p. for 5 days) produced a significant increase in hepatic cytochrome P-450, aminopyrine N-demethylase and benzphetamine N-demethylase activities, but not in the rest of the aforementioned parameters of biotransformation reactions. These data indicate that the effect of garlic oil on the hepatic drug-metabolizing enzyme system is dose-dependent.

Allyl Compounds↗
PubMed1991

Effect of microsomal enzyme inducing agents on hepatic biotransformation in cotton rats (Sigmodon hispidus): comparison to that in Sprague-Dawley rats.

1. Activities of several biotransformation enzymes were determined in male and female Sigmodon hispidus. Benzphetamine N-demethylase and glutathione S-transferases toward 1-chloro-2,4-dinitrobenzene and sulfobromophthalein were higher in male Sigmodon hispidus than the female animals. 2. The study also determined the effect of microsomal enzyme inducing agents on hepatic biotransformation in male Sigmodon hispidus. 3. Cytochrome P-450 concentration was similar in cotton and Sprague-Dawley rats, and was increased after phenobarbital, pregnenolone-16 alpha-carbonitrile, or 3-methylcholanthrene treatment. 4. Benzphetamine N-demethylase was 4-fold higher in Sigmodon hispidus and was induced by 75-100% after phenobarbital. 5. UDP-Glucuronosyltransferase toward estrone, 1-naphthol, diethylstilbestrol and testosterone was 2- to 4-fold higher in cotton rats and was not altered by treatment with the inducing agents. 6. Conjugation of 1-chloro-2,4-dinitrobenzene, ethacrynic acid and sulfobromophthalein with glutathione was similar in both rodent species and was not inducible. 7. Sulfation of 2-naphthol was 15-30% of that in Sprague-Dawley rats and was not increased by inducer administration.

Animals↗
PubMed1993

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-ethoxycoumarin 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.

7-Alkoxycoumarin O-Dealkylase↗
PubMed2005

Role of cytochrome b5 in catalysis by cytochrome P450 2B4.

Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of cytochrome b5 in cytochrome P450 catalysis using two substrates, methoxyflurane and benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b5. The other is benzphetamine, whose metabolism is minimally modified by cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both cytochrome b5 and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.

Animals↗
PubMed2002

Molecular cloning and characterization of mouse CYP2J6, an unstable cytochrome P450 isoform.

A cDNA encoding a new cytochrome P450 was cloned from a mouse liver library. Sequence analysis revealed that this 2046-bp cDNA encodes a 501-amino acid polypeptide that is 72-94% identical to other CYP2J subfamily P450s and is designated CYP2J6. Northern analysis demonstrated that CYP2J6 transcripts are abundant in the small intestine and present at lower levels in other mouse tissues. In situ hybridization revealed that CYP2J6 mRNAs are present in luminal epithelial cells of the gastrointestinal mucosa. The CYP2J6 cDNA was expressed in Sf9 cells using baculovirus. The heterologously expressed CYP2J6 protein displayed a typical P450 CO-difference spectrum; however, the protein was unstable as evidenced by the loss of the Soret maxima at 450nm and the appearance of a 420nm peak when CYP2J6-expressing cells were disrupted by mechanical homogenization, sonication, or freeze-thaw. Immunoblotting of mouse microsomes with the anti-human CYP2J2 IgG, which cross-reacts with rodent CYP2Js, demonstrated the presence of multiple distinct murine CYP2J immunoreactive proteins in various tissues. Immunoblotting with an antibody to a CYP2J6-specific peptide detected a prominent 55-57kDa protein in Sf9 cell extracts expressing recombinant CYP2J6 but did not detect a protein of similar molecular mass in mouse small intestinal microsomes. Mixing experiments demonstrated that recombinant CYP2J6 is degraded rapidly in the presence of small intestinal microsomes consistent with proteolysis at highly sensitive sites. Sf9 cells, which express both CYP2J6 and NADPH-P450 oxidoreductase, metabolized benzphetamine but not arachidonic acid. We conclude that CYP2J6 is an unstable P450 that is active in the metabolism of benzphetamine, but not arachidonic acid.

Amino Acid Sequence↗
PubMed1998

Consequences of overexpression of growth hormone in transgenic mice on liver cytochrome P450 enzymes.

The effect of growth hormone (GH) on cytochrome P450 (CYP) and P450-dependent monooxygenases was studied in 4-, 6-, 8-, and 10-month-old female bovine growth hormone (bGH) transgenic mice that overexpress GH. Nontransgenic female mice (C57/SJL) littermates were used for baseline determinations. The body weights of the bGH mice were approximately 35% greater than those of the controls. The liver weights were 2-fold higher than those of the controls, resulting in a 25-60% increase in liver/body weight ratio during the life span of the bGH mice when compared with the controls. Similar increases in heart and kidney weights were observed. Since the GH transgene was transcriptionally regulated by a metallothionein-I gene promoter, metallothionein concentrations in livers of transgenic and nontransgenic mice were measured. No significant differences were observed. In marked contrast to increases in liver weights, hepatic cytochrome P450 content, benzphetamine N-demethylase, and benzo [a] pyrene hydroxylase activities were decreased by 36, 42 and 75%, respectively. No age-related changes in the decrease of the monooxygenases were observed. Microsomal heme oxygenase (HO) in the liver was induced 44% above the control values. Immunoblot analysis also showed a marked increase in HO-1 in the bGH mice. These results indicate that GH suppresses the carcinogen-metabolizing enzyme benzo [a] pyrene hydroxylase and the drug-metabolizing enzyme benzphetamine N-demethylase. This suppression was accompanied by an induction of HO activity in bGH transgenic mice. The consequences of prolonged exposure to supraphysiological levels of this hormone cannot always be predicted from the known physiological actions of GH.

Animals↗
PubMed1987

A selective enhancement of xenobiotic metabolizing systems of rat lungs by prolonged exposure to ozone.

Male Wistar rats were exposed to 0.2 and 0.4 ppm O3 for 4, 8, and 12 weeks and to 0.1 and 0.2 ppm O3 for 4 weeks to examine the effects of prolonged exposure to O3 on the xenobiotic metabolizing systems in the lung. Exposures to 0.2 and 0.4 ppm O3 caused a significant increase in the NADPH-cytochrome P-450 reductase activity and cytochrome P-450 content in a dose-dependent manner during 4-12 weeks, whereas NADH-cytochrome b5 reductase was not altered. After 12 weeks of exposure to 0.4 ppm O3, NADPH-cytochrome P-450 reductase and cytochrome P-450 reached a maximum showing 138 (P less than 0.001) and 221% (P less than 0.001) those of the control levels, respectively. At 0.1 ppm O3, the cytochrome P-450 content still increased significantly to 152% that of the control on the fourth week. In parallel to the increment of cytochrome P-450 benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase increased significantly during 4-12 weeks of exposures to 0.2 and 0.4 ppm O3. After a 4-week exposure to 0.1 and 0.2 ppm O3, benzphetamine N-demethylase increased to the largest degree among the enzymes metabolizing four kinds of xenobiotics examined. 7-Ethoxycoumarin O-deethylase also increased significantly but of smaller magnitude, whereas coumarin hydroxylase was not affected. These results show that prolonged exposures to 0.1-0.4 ppm O3 persistently enhance pulmonary cytochrome P-450 systems. It is also suggested that some isozyme(s) of pulmonary cytochrome P-450, especially that catalyzing benzphetamine N-demethylation, are preferentially increased by exposure to low levels of O3.

Animals↗
PubMed2002

Mixed function oxidases in kidney and duodenum of camel, guinea pig and rat.

The activities of the drug-metabolizing enzymes, aniline 4-hydroxylase, benzphetamine N-demethylase and 7-ethoxycoumarin O-deethylase have been measured in vitro in kidneys and duodenum of camels (Camelus dromedarius), guinea pigs (Cavia porcellus) and rats (Rattus norvegicus). In these species, levels of hepatic microsomal parameters namely microsomal protein, cytochrome P(450), cytochrome b(5) and NADPH-cytochrome c reductase have also been determined. In general, camels seemed to have the lowest enzyme activity when compared to rats and guinea pigs. Rats showed the highest activity in NADPH-cytochrome c reductase, aniline 4-hydroxylase and ethoxycoumarin O-deethylase among these species. However, guinea pigs showed the highest enzyme activity in cytochrome P(450), cytochrome b(5) and benzphetamine N-demethylase.

Animals↗
PubMed1995

Substrate-dependent competition of different P450 isozymes for limiting NADPH-cytochrome P450 reductase.

The goal of these studies was to demonstrate that one P450 isozyme can influence the function of another isozyme when combined in a reconstituted system. Benzphetamine and 7-pentoxyresorufin were both shown to be preferred substrates for P450 2B4 (LM2) as compared to P450 1A2 (LM4). However, these substrates exhibited different characteristics when examined in reconstituted systems containing reductase and both P450 isozymes. Whereas benzphetamine demethylation showed a small increase in catalytic activity when both P450 1A2 and 2B4 were present over the activities obtained in simple reconstituted systems, 7-pentoxyresorufin O-dealkylation (PROD) was dramatically inhibited when both isozymes were present. These results indicate that the functional interactions between P450s in complex reconstituted systems are dependent on the substrate present. Inhibition of PROD was also dependent on reductase levels, with the inhibitory effect being more pronounced at subsaturating reductase. Finally, these protein-protein interactions were shown to be dependent on the reductase concentration in the reconstituted system rather the P450 concentration, supporting the view that P450 1A2 is inhibiting the reaction by competing with P450 2B4 for reductase molecules.

Animals↗
PubMed1994

Photoaffinity labeling of cytochrome P450 2B4: capture of active site heme ligands by a photocarbene.

Spiro[adamantane-2,2'-diazirine], which produces adamantyl carbene upon photolysis, binds tightly to P450 2B4 (KS = 3.2 microM), giving a normal substrate binding difference spectrum. Irradiation of 2-[3H]adamantane diazirine at 365 nm in the presence of native, ferric P450 2B4 resulted in first-order photolysis (t1/2 = 1.8 min). The main product was 2-[3H]adamantanol, with about 6% of the radioactivity covalently bound to P450 2B4. With the ferrous carbonyl form of P450 2B4, 2-adamantanol production decreased and protein labeling increased to 12%. When ferric cyanide 2B4 was used, 2-adamantanecarbonitrile was formed in addition to 2-adamantanol. The nitrile appears to have resulted from capture of the iron-bound cyanide ligand by the carbene. The use of multiple cycles of photolysis increased the percentage of protein labeling to 76%. Photolabeling was inhibited by known 2B4 substrates and inhibitors. Also, N-demethylation of benzphetamine and generation of a substrate binding difference spectrum by benzphetamine were both inhibited stoichiometrically with the fraction of radiolabeled protein. The labeled protein was permanently converted to the high-spin state, as indicated by the characteristic change in the absorbance spectrum, demonstrating irreversible occupation of the substrate binding site by the adamantyl residue. Mild acid hydrolysis of radiolabeled 2B4 at the five Asp-Pro bonds generated a 2-kDa peptide which carried 78% of the radioactivity. These results are interpreted as the result of the active site carbene reacting by three competing pathways: capture of the heme sixth ligand to yield either 2-adamantanol or 2-adamantanecarbonitrile, capture of an unbound active site water molecule to yield adamantanol, and covalent attachment to a protein residue. Thus, the P450 2B4 active site appears to contain at least one unbound water molecule in addition to the heme aquo sixth ligand, even when substrate is present.

Adamantane↗