Mixed-function oxidations, cytochrome

Estabrook, R.W., Hildebrandt, A.G., Baron, J., Netter, K.J. and Leibman, K. (1971) A new spectral intermediate associated with cytochrome P-450 function in liver microsomes. Biochemical and Biophysical Research Communications, 42 (1), 132-139. Pompon, D. and Coon, M.J. (1984) On the mechanism of action of cytochrome P-450. Oxidation and reduction of the ferrous dioxygen complex of liver microsomal cytochrome P-450 by cytochrome b5. Journal of Biological Chemistry, 259 (24), 15377-15385. Hildebrandt, A. and Estabrook, R.W. (1971) Evidence for the participation of cytochrome b 5 in hepatic microsomal mixed-function oxidation reactions. Archives of Biochemistry and Biophysics, 143 (1), 66-79. 

Franklin R. Inhibition of mixed-function oxidations by substrates forming reduced cytochrome P-450 metabolic-intermediate complexes. Pharmacol Ther 1977 2 227-245. 

Microsomes contain, in addition to the two cytochrome reductases just discussed, a flavoprotein which catalyzes the mixed function oxidation of secondary and tertiary amines to the hydroxylamines and amine oxides, respectively (333, 334). This flavoprotein, which contains about 2 moles of phospholipid and 1 mole of FAD per 70,000 g of protein, is specific for NADPH (333, 334) The enzyme is also able to catalyze the further oxidation of the hydroxylamines to nitrones (336). The reactions... 

Lipoxygenase Malate dehydrogenase Metallo-endopeptidase N-Methyl transferase Monoamine oxidase Mixed-function oxidase (cytochrome P450 dependent) NADH2 diaphorase NADPH2 diaphorase Neutral endopeptidase 24.11 Nitro oxide synthase Nitro reductase 5 -Nucleotidase Peroxidase... 

In silico estimation of metabolism is still an area of intense study and development. Accurate prediction of intrinsic clearance is still not possible with the currently available methods [15]. Most of the progress in this area has been focused on the mixed function oxidase cytochrome P450 enzyme family. Advances in this area have been focused on three areas (1) prediction of the cytochrome P450 (CYP) enzyme isotype that is responsible for the major metabolism, (2) prediction of the chemical site of a molecule that is most likely to undergo biotransformation by oxidative metabolism, and (3) structure-based docking studies of CYP enzyme substrate complexes. ... 

This step is a mixed-function oxidation reaction similar to the one catalyzed by cytochrome P-450 reductase and there is considerable homology between NOS and cytochrome P-450 reductase. In the second step, further oxidation of N -hydroxyl arginine yields NO and citrulline ... 

The microsomal fraction of liver contains a monooxygenase system consisting of cytochrome P-450 (EC 1.14.14.1), NADPH-cytochrome P-450 reductase (EC 1.6.2.4), and phospholipid. This system catalyzes the hydroxylation of a large number of both foreign and endogenous compounds in a mixed-function oxidation reaction using molecular oxygen and NADPH or NADH as electron donors ... 

Hildebrandt, A. and R.W. Estabrook (1971). Evidence for the participation of cytochrome b5 in hepatic microsomal mixed-function oxidation reactions. Arch. Biochem. Biophys. 143, 66-79. 

The fact that cytochrome P-450 functions as terminal oxidase for these reactions is supported by both indirect and direct evidence (a) the drug-induced increase in the hydroxylase activity of liver microsomes is accompanied by a similar increase in microsomal cytochrome P-450 (25, 26), an indication that this pigment participates in the mixed-function oxidation of drugs (b) maximal reversal of the characteristic CO inhibition of mixed function oxidations is accomplished by illuminating the enzyme assay system with monochromatic light of 450 m/x wavelength, the absorption maximum of the CO compound of reduced cytochrome P-450 (P-450 -CO). 

Thus, the reduced form of cytochrome P-450 functions as the oxygenactivating biocatalyst of a wide variety of mixed-function oxidations by vertebrate tissues effecting biosynthesis and catabolism of steroid hormones, bile acid formation, and the metabolism of drugs and other xeno-biotics (16). Since reduced pyridine nucleotides do not react directly with hemoproteins, the hydroxylase systems must include components that mediate the electron transport from TPNH to cytochrome P-450. There also must be distinctive diflFerences in composition causing the substrate specificity of the oxygenations. 

The experimental results given above clearly indicate that cytochrome P-450 is the oxygen-activating catalyst of a wide variety of mixed-function oxidations in tissues of vertebrate animals. Spectrophoto-metric measurements and determinations of the photochemical action spectra did not reveal any significant differences between the pigments from different sources. Yet the substrate specificity of these mixed-function oxidase systems is pronounced, as shown by studies on the induction of microsomal hydroxylase activity toward different xenobiotics (27) and on the affinity of different C-21 methyl corticosteroids toward... 

Zantac gives rise to three known metabolites, namely (a) ranitidine N-oxide (Z ) ranitidine S-oxide and (c) desmethyl ranitidine. It is observed to be only a weak inhibitor of the hepatic cytochrome P-450 mixed function oxidation system. The plasma half-life ranges between 2 to 3 hours and it usually gets excreted together with its metabolites in the urine. 

James MO, Little PJ (1980) Characterization of cytochrome P-450 dependent mixed-function oxidation in the spiny lobster, Panulirus argus. In Gustafsson JA, Carlstedt-Duke J, Mode A, Rafter J (eds) Biochemistry, biophysics and regulation of cytochrome P-450. Elsevier/North-Holland, Amsterdam, pp 113-120... 

Lipscomb, J. D. (1974) Energy transfer and segregation mixed function oxidation by cytochrome P450otm and putidaredoxin. Ph.D. Thesis, Universit) of Illinois. 

Oxidation is intimately linked to the activation of polycyclic aromatic hydrocarbons (PAH) to carcinogens (1-3). Oxidation of PAH in animals and man is enzyme-catalyzed and is a response to the introduction of foreign compounds into the cellular environment. The most intensively studied enzyme of PAH oxidation is cytochrome P-450, which is a mixed-function oxidase that receives its electrons from NADPH via a one or two component electron transport chain (10. Some forms of this enzyme play a major role in systemic metabolism of PAH (4 ). However, there are numerous examples of carcinogens that require metabolic activation, including PAH, that induce cancer in tissues with low mixed-function oxidase activity ( 5). In order to comprehensively evaluate the metabolic activation of PAH, one must consider all cellular pathways for their oxidative activation. 

A potentially powerful probe for sorting out the contribution of hydroperoxide-dependent and mixed-function oxidase-dependent polycyclic hydrocarbon oxidation is stereochemistry. Figure 9 summarizes the stereochemical differences in epoxidation of ( )-BP-7,8-dihydrodiol by hydroperoxide-dependent and mixed-function oxidase-dependent pathways (31,55,56). The (-)-enantiomer of BP-7,8-dihydrodiol is converted primarily to the (+)-anti-diol epoxide by both pathways whereas the (+)-enantiomer of BP-7,8-dihydrodiol is converted primarily to the (-)-anti-diol epoxide by hydroperoxide-dependent oxidation and to the (+)-syn-diol epoxide by mixed-function oxidases. The stereochemical course of oxidation by cytochrome P-450 isoenzymes was first elucidated for the methycholanthrene-inducible form but we have detected the same stereochemical profile using rat liver microsomes from control, phenobarbital-, or methyl-cholanthrene-induced animals (32). The only difference between the microsomal preparations is the rate of oxidation. 

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