The cytochrome P-450 Substrate optical difference spectra of pesticides with mouse hepatic microsomes (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1973
Phenobarbital and naphthalene induce a new species of cytochrome P-450 in houseflies which has a maximum absorbance in the off balance absolute spectrum at 446 nm. 2. Neither induced nor non-induced hemoprotein show type I spectral changes in the oxidized state but only when they are reduced by NADPH, Na2S204 or when ethyl isocyanide is present in the reaction mixture. The affinity of the induced hemoprotein for type I and II substrates is greater than in the case of control cytochrome P-450. 3. As in mammalian systems, the type I spectral changes depend on the type I substrate concentration. However, control cytochrome P-450 shows small spectral changes at low substrate concentrations, and only at concentrations above 3 mM the spectral changes become large. On the contrary, appreciable type I spectral changes can be observed in the case of induced hemoprotein with substrate concentrations as low as 0.05 mM. This is particularly apparent in the case of induced hemoprotein previously incubated with ethyl isocyanide. Although the extinction coefficient for the Soret peaks produced by ethyl isocyanide appears to be the same for control and induced hemoprotein, the titration of spectral changes for the 455-nm peak is different in control and induced cytochrome P-450. 4. It is suggested that chemical changes involving the heme moiety of cytochrome P-450 bring about conformational changes in the protein moiety which facilitate the binding of type I ligands. Whether this phenomenon has an allosteric nature remains to be established. Abbreviation: SKF 252-A, 2-diethyl aminoethyl diphenyl propyl acetate. * In partial fulfillment of the requirements of the University of Georgia Graduate School for a Ph.D. degree (J.C.
Spectra and Kinetic Studies of the Compound I Derivative of Cytochrome P450 119
Journal of the American Chemical Society, 2008
The Compound I derivative of cytochrome P450 119 (CYP119) was produced by laser flash photolysis of the corresponding Compound II derivative, which, in turn, was prepared by reaction of the resting enzyme with peroxynitrite. The UV-visible spectrum of the Compound I species contains an asymmetric Soret band that can be resolved into overlapping transitions centered at ca. 367 and 416 nm and a Q-band with λ max ≈ 650 nm. Reactions of the Compound I derivative with organic substrates gave epoxideized (alkene oxidations) and hydroxylated (C-H oxidations) products as demonstrated by product studies and oxygen-18 labeling studies. The kinetics of oxidations by CYP119 Compound I were measured directly; the reactions included hydroxylations of benzyl alcohol, ethylbenzene, Tris buffer, lauric acid, and methyl laurate, and epoxidations of styrene and 10-undecenoic acid. Apparent second-order rate constants, equal to the product of the equilibrium binding constant (K bind) times the first-order oxidation rate constant (k ox), were obtained for all substrates. The oxidations of lauric acid and methyl laurate displayed saturation kinetic behavior, which permitted solution of both K bind and k ox for these substrates. The unactivated C-H positions of lauric acid reacted with a rate constant of k ox = 0.8 s −1 at room temperature. The CYP119 Compound I derivative is more reactive than model Compound I species, iron(IV)-oxo porphyrin radical cations, and similar in reactivity to the Compound I derivative of the heme-thiolate enzyme chloroperoxidase. Kinetic isotope effects (k H /k D) for oxidations of benzyl alcohol and ethylbenzene were small, reflecting the increased reactivity of the Compound I derivative in comparison to models. Nonetheless, CYP119 Compound I apparently is much less reactive than the oxidizing species formed in the P450 cam reaction cycle. Competition kinetic studies employing CYP119 activated by hydrogen peroxide indicate that the same oxidizing transient is formed in the photochemical reaction and in the hydrogen peroxide shunt reaction. The ubiquitous cytochrome P450 (CYP or P450) enzymes are heme-containing enzymes with thiolate from protein cysteine as the fifth ligand to iron. 1 P450s serve in several catalytic roles in nature, but the major function is to catalyze oxidation reactions, typically via two-electron, oxo-transfer processes. In Man, the P450s perform both highly specific reactions, such as oxidation of androgens to estrogens, and broad-spectrum oxidations of drugs, pro-drugs, and xenobiotics in the liver. 2 Much of the interest in P450s derives from the pharmaceutical impact of these enzymes and their relationships to disease states, including cancers and liver disease, that result from over-expression of P450s. 3-5
A model compound study of the CO-adduct of cytochrome P-450
The Journal of biological chemistry, 1974
A model compound was prepared having the same optical properties in the Soret and visible regions as the CO-adduct of ferrous cytochrome P-450. Obligate for the formation of this spectral species absorbing at 450 nm (emM = 93) and 555 nm (emM = 23) in a dimethylsulfoxide-ethanol solvent system was the presence of ferrous heme, CO, thiol, and a strong base. Decreasing the dielectric constant of the solvent, which would be equivalent to increasing the hydrophobicity of the heme environment in the protein, causes a small shift in the Soret absorption to the blue with no change m emM. The absence of thiol or strong base resulted in the formation of typical heme. CO absorptions at 413 nm (emM = 150), 535 nm (emM = 25), and 566 nm (emM = 30). Jt is concluded that the sulfur-containing axial ligand in the model compound and also in ferrous cytochromes P-450. CO is a mercaptide anion. Cytochromes P-450 form a class of unique hemoproteins, members of which participate in the mixed function oxidation of a wide variety of substrates (l-4). These hemoproteins derive their name, in part, from the atypical Soret absorption maximum they exhibit near 450 nm (5-7) when reduced and ligated with carbon monoxide. All other hemoproteins capable of binding CO, including cytochromes P-420, which are denatured forms of cytochromes P-450 (8), demonstrate an absorption maximum near 420 nm when reduced and ligated to CO. The identification of the unusual axial ligands of cytochrome P-450 is essential to our understanding enzymic function, especially since oxygen is presumed to bind to the heme (9). Since CO also binds to the heme, produces the unusual Soret, and inhibits enzymic function (lo), it would be useful to ascribe
Chemical Research in Toxicology, 2013
A total of 68 chemicals including derivatives of naphthalene, phenanthrene, fluoranthene, pyrene, biphenyl, and flavone were examined for their abilities to interact with human P450s 2A13 and 2A6. Fifty-one of these 68 chemicals induced stronger Type I binding spectra (iron low-to highspin state shift) with P450 2A13 than those seen with P450 2A6, i.e. the spectral binding intensities (ΔA max /K s ratio) determined with these chemicals were always higher for P450 2A13. In addition, benzo[c]phenanthrene, fluoranthene, 2,3-dihydroxy-2,3-dihydrofluoranthene, pyrene, 1-hydroxypyrene, 1-nitropyrene, 1-acetylpyrene, 2-acetylpyrene, 2,5,2',5'-tetrachlorobiphenyl, 7hydroxyflavone, chrysin, and galangin were found to induce a Type I spectral change only with P450 2A13. Coumarin 7-hydroxylation, catalyzed by P450 2A13, was strongly inhibited by 2'methoxy-5,7-dihydroxyflavone, 2-ethynylnaphthalene, 2'-methoxyflavone, 2-naphththalene propargyl ether, acenaphthene, acenaphthylene, naphthalene, 1-acetylpyrene, flavanone, chrysin, 3-ethynylphenanthrene, flavone, and 7-hydroxyflavone; these chemicals induced Type I spectral changes with low K s values. On the basis of the intensities of the spectral changes and inhibition of P450 2A13, we classified the 68 chemicals into eight groups based on the order of affinities for these chemicals and inhibition of P450 2A13. The metabolism of chemicals by P450 2A13 during the assays explained why some of the chemicals that bound well were poor inhibitors of P450 2A13. Finally, we compared the 68 chemicals for their abilities to induce Type I spectral changes of P450 2A13 with the Reverse Type I binding spectra observed with P450 1B1: 45 chemicals
Biochemical …, 1985
The individual members of a homologous series of phenoxazone ethers related to ethoxyresorufin were 0-dealkylated, and the parent compound phenoxazone was ring-hydroxylated, each at different rates with hepatic microsomes of untreated rats. A structure-activity relationship (SAR) was plotted, relating the rate of 0-deaikylation to the length and type of the ether side-chain. Phenobarbitone (PB), 3-methylchoIanthrene (MC), Aroclor 1254 (ARO), isosafrole (ISO) and SKF-525A each induced preferentially the O-dealkylation of different members of the homologous series, resulting in the appearance of 5 different SAR plots, which characterized and differentiated between the 5 different inducers. BNapthoflavone (BNF) had a similar effect to MC, whereas pregnenolone 16cu-carbonitrile treatment caused no large change in the metabolism of any of the substrates tested. For characterizing the effects of the different inducers it was largely sufficient to compare the 0-dealkylations of just 4 of the ethers: methoxy-, ethoxy-, pentoxy-and benzyloxyphenoxazone.
Studies on the substrate-binding sites of liver microsomal cytochrome P -448
Biochemical Journal, 1982
The interaction of substrates of the microsomal mixed-function oxidases with cytochromes P-450 and P-448 was investigated by using liver microsomes from rats pretreated with phenobarbital or 3-methylcholanthrene, and with purified forms of the cytochromes isolated from rabbit liver. The two forms of the cytochrome have different substrate specificities; cytochrome P-450 has one type 1 substrate-binding site that can accommodate a large variety of substrates, but in contrast cytochrome P-448 may possess two type 1 substrate-binding sites, one of which is different to that of cytochrome P-450 in that it shows a specificity for substrates such as safrole and 9-hydroxy-ellipticine. These findings explain why the two forms of the cytochrome have different substrate specificities and play contrasting roles in the activation and deactivation of xenobiotics.
European journal of biochemistry / FEBS, 1978
The mechanism of liver microsomal aromatic hydroxylation has been investigated by using cumene hydroperoxide as the hydroxylating agent and comparing this reaction with the NADPH-dependent reaction. The conversion of [4-(3)H]acetanilide to 4-hydroxyacetanilide by rat liver microsomes (or purified cytochrome P-450) in the presence of either cumene hydroperoxide or NADPH is attended by comparable 'NIH shifts'. This indicates that hydroxylation in the two systems proceeds via a common intermediate, presumably an arene oxide. The intermediacy of an arene oxide, phenanthrene-9,10-oxide, is established by incubating [3-(3)H]-phenanthrene with rat-liver microsomes and cumene hydroperoxide in the presence of either non-radioactive phenanthrene-9,10-oxide as a 'trap' or in the presence of cyclohexene oxide, an inhibitor of the enzyme epoxide hydrase. Incubation of phenanthrene with cumene hydroperoxide in an 18O-enriched medium has confirmed that the oxygen atom in phenanthre...