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Publisher Summary This chapter discusses the N-terminal modifications that alter P450 membrane targeting and function. The hydrophobic N-terminal region that is present in mammalian P450 cytochromes serves as the signal peptide for cotranslational insertion of the protein into the endoplasmic reticulum, while the following hydrophobic proline-rich segment promotes stabilization of the enzyme. The role of the N-terminal region in promoting extensive aggregation of P450 enzymes is demonstrated by disaggregation of shortened P450s to monomers by the detergent cholate at concentrations that do not alter the multimeric aggregation state of the full-length enzymes. The retention of catalytic activity and the monomerization of P450 cytochromes at low detergent concentrations, achieved through N-terminal modification, are used to improve the suitability of these enzymes as candidates for crystallization attempts. The chapter describes the methods for the heterologous expression, determination of subcellular localization, and purification of shortened P450 cytochromes as well as examination of their catalytic properties.

Publisher Summary This chapter discusses the purification and characterization of cytochromes P450 in rabbit nasal microsomes. The nasal mucosa is rich in P450 cytochromes and other biotransformation enzymes, with the specific content of P450 in nasal microsomes being the highest among all rabbit extrahepatic tissues examined. A major portion of the microsomal P450 in rabbit olfactory mucosa is made up of two unique forms, termed P450 NMa and NMb, and the nasal tissues also contain P450 forms that are similar to the isozymes previously found in liver, such as P450 form 3a. The method described in this chapter for the isolation of NMa and NMb, which are the only nasal P450 cytochromes purified and characterized to date. NMa is present in both olfactory and respiratory mucosa, and in liver, whereas NMb is detected only in olfactory mucosa. Neither form is found in other tissues, including brain, lung, heart, intestinal mucosa, kidney, and esophageal mucosa.

Publisher Summary This chapter describes the reductive cleavage of hydroperoxides by cytochrome P- 450. Various forms of liver microsomal cytochrome P-450 (cytochrome P- 450 LM ) are known to catalyze numerous chemical reactions, including the hydroxylation of fatty acids, steroids, and a variety of foreign compounds, such as drugs and carcinogens. These monooxygenation reactions require aerobic conditions and nicotine adenine dinucleotide phosphate hydrogenase (NADPH) as the electron donor in microsomal suspensions or in the reconstituted system containing purified cytochrome P-450 LM , purified NADPH-cytochrome-P-450 reductase, and phosphatidylcholine. On the other hand, molecular oxygen, NADPH, and the reductase are not required when organic hydroperoxides or related compounds serve as the oxygen donor in cytochrome P-450 LM -catalyzed substrate hydroxylations. In such reactions, the hydroperoxides are converted to the corresponding alcohols so that cumyl hydroperoxide, for example, is converted to cumyl alcohol. Other model hydroperoxides in addition to cumyl serve as substrates in the reductive cleavage reaction. α-Methylbenzyl hydroperoxide yields acetophenone as well as benzaldehyde because either a hydrogen atom or a methyl group can be lost, benzyl hydroperoxide yields benzaldehyde, and tert -butyl hydroperoxide yields acetone and methane. With limiting amounts of cytochrome P-450 form present, the turnover numbers, expressed as nanomoles of NADPH oxidized per minute per nanomole of the cytochrome, are 60, 30, 24, and 6 for tert -butyl, cumyl, α-methylbenzyl, and benzyl hydroperoxides, respectively. Pentane has been identified as a cleavage product of the 15-hydroperoxide derived from arachidonic acid as well as from the 13-hydroperoxide derived from linoleic acid.

Publisher Summary This chapter discusses assays for cytochrome P- 450 peroxygenase activity. Toluene, which yields benzyl alcohol in the cytochrome P- 450-catalyzed peroxygenase reaction, is a useful substrate in mechanistic studies. To determine benzyl alcohol formation at fixed time intervals, a gas chromatographic assay is employed. Cytochrome P- 450 is versatile enzyme that catalyzes numerous types of chemical reactions with a variety of substrates, including the hydroxylation of xenobiotics, such as drugs, pesticides, dyes, organic solvents, anesthetics, and carcinogens, as well as naturally occurring lipids, including steroids, fatty acids, and prostaglandins. Molecular oxygen serves as the natural donor when electrons are supplied to cytochrome P- 450 by nicotinamide adenine dinucleotide phosphate (NADPH) via proteins that serve as electron carriers. In the case of liver microsomes or the reconstituted enzyme system, NADPH-cytochrome- P- 450 reductase serves as the carrier and in the overall reaction equimolar amounts of substrate, O 2 , and NADPH are consumed and equimolar amounts of hydroxylated substrate, H 2 O, and NADP are formed. Molecular oxygen can be replaced by a variety of hydroperoxides and related artificial donors. Following the discovery of the ability of cytochrome P- 450-containing microsomal suspensions to promote the oxygenation of an organic substrate at the expense of an alkyl hydroperoxide, other oxidants, such as peroxy acids, periodate, iodosobenzene, iodobenzene diacetate, and N-oxides, are found to function in a similar capacity.

Publisher Summary Cytochrome P-450 was first recognized as a pigment in liver microsomes from the absorption peak of its carbon monoxide complex at 450 nm. This CO-binding pigment is found to be a cytochrome of the b type, which is reducible by both NADPH and NADH under anaerobic conditions and is rapidly reoxidizable in the presence of molecular oxygen. The photochemical action spectrum for the reversal of carbon monoxide inhibition of steroid and drug hydroxylation was shown to have a maximum at 450 nm. In contrast to the hepatic cytochromes, which are generally not substrate-specific, the P-450's found in microsomes and mitochondria from endocrine tissues have a much greater selectivity in steroid transformations. The enzyme is also found in plants and in microorganisms, the best studied example being P -450 cam , the cytochrome induced by growth of Pseudomonas putida on camphor. P -450 cam , which is obtained in a monomeric, soluble state from this organism, has been purified to homogeneity, crystallized, and thoroughly characterized. In the microsomal systems, electron transfer occurs directly from NADPH-cytochrome P-450 reductase to the cytochrome, whereas in the mitochondria1and bacterial systems the reductase contains a single flavin group, and an iron-sulfur protein serves as the direct donor. The P-450's that are involved in the biosynthesis of steroid hormones, bile acids, and the active forms of vitamin D 3 exhibit a high degree of substrate and regiospecificity. The steroid hormone-synthesizing enzymes involved in the production of mammalian hormones are of both mitochondria1 and microsomal origin and are distributed in various tissues—including adrenal cortex, ovary, testis, and placenta.

Publisher Summary This chapter presents a few studies in which purified forms of the cytochrome from rabbit liver microsomes was used. The ability of rabbit liver microsomal cytochrome P-450 to catalyze the oxidation of ethanol to acetaldehyde was examined in a reconstituted enzyme system containing electrophoretically homogenous phenobarbital-inducible P-450LM2, β-naphtoflavone-inducible P-450LM4, or a partially purified mixture of P-450LM1, and LM7. Only very low rates of ethanol oxidation were observed, about 3–5 nmol per min per nmol P-450LM, with no apparent specificity of any of the P-450LM forms for ethanol. In the presence of ethanol, essentially all of the NADPH oxidation in the reconstituted system could be accounted for by the formation of H2O2 finding. The inability of ethanol to stimulate NADPH and oxygen utilization by the reconstituted enzyme system are incompatible with a mixed function oxidation reaction leading to acetaldehyde formation. Ethanol had no effect on benzphetamine hydroxylation by P-450LM2 rates of ethanol oxidation could possibly be because of a slight peroxidatic activity of P-450LM in the presence of H2O2 generated in the reconstituted system.

When reduced with NADPH under anaerobic conditions, FMN-depleted NADPH-cytochrome P-450 reductase does not catalyze rapid electron transfer to P-450 LM . This activity is restored when the enzyme is reconstituted with FMN or a variety of artificial flavins, with the exception of 5-deaza-FMN. It was also found that the rate of P-450 LM 4 reduction by flavoprotein containing functional analogues such as 8-C1- and iso-FMN differs from that of the native enzyme. These and other results are consistent with a role for FMN in the transfer of electrons to oxidized P-450 LM via the reaction, FMNH 2 → FMNH.

The metabolism of benzo[a]pyrene (BP) and of the (−) and (+) enantiomers of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (trans-7,8-diol) were examined in a reconstituted enzyme system containing rabbit phenobarbital-inducible P-450LM2 or 5,6-benzoflavone-inducible P-450LM4; the metabolites formed were analyzed by HPLC. The two cytochrome preparations exhibit large qualitative and quantitative differences with these substrates. Based on total products, P-450LM2 oxygenates BP ten-fold faster than P-450LM4. Phenols comprise 86% of the BP metabolites with P-450*LM2 and only 55% of those with P-450LM4, whereas quinones constitute 12 and 45% of the metabolites with P-450LM2 and LM4, respectively. The relative amounts of the two phenol peaks are different for the two cytochromes, P-450LM2 showing a somewhat greater regioselectivity for the 3 position than P-450LM4. The specificity for trans-7,8-diol in the reconstituted enzyme system resides in P-450LM4 since, in contrast to its poor catalytic activity toward BP, this cytochrome is 15 to 30 times more active with the (−) enantiomer and 4 to 7 times more active with the (+) enantiomer of the diol than P-450LM2. Both P-450LM4 and LM2 display higher activities with (−) relative to the (+) optical isomer of the substrate, but this difference is most striking with P-450LM4 (activity ratio (−)/(+) = 10). P-450LM4 oxygenates (−) trans-7,8-diol predominantly to diol-epoxide I (product ratio I/II =11), whereas about twice as much diol-epoxide II as I is produced with (+)-trans-7,8-diol as substrate. P-450LM2 is devoid of product stereospecificity, for it yields about equal amounts of diol-epoxide I and II with either enantiomer of the trans-7,8-diol. These results indicate that BP and trans-7,8-diol metabolism are catalyzed by different isozymes of rabbit liver microsomal cytochrome P-450 and that P-450LM4 possesses a pronounced stereospecificity since it inserts the oxygen atom primarily on one side of the plane of the trans-7,8-diol molecule, independent of the configuration of the 7,8 hydroxyl groups. Thus, regio- and stereospecificities of different P-450LM isozymes may be important factors in determining the biological effects of BP.

Trypsinolysis of purified rabbit liver microsomal NADPH-cytochrome P-450 reductase yields a large, soluble fragment capable of reducing cytochrome c but not P-450 LM , and a small fragment or “tail peptide” which has been isolated and shown by its solubility and amino acid composition to be hydrophobic in nature. The large fragment is unable to bind to the cytochrome, as shown by gel exclusion chromatography and difference spectrophotometry. These and other experiments indicate that the hydrophobic tail peptide is required for formation of a functional complex between the reductase and P-450 LM in the reconstituted hydroxylation system.

Publisher Summary This chapter describes the method for the isolation of two homogeneous forms, phenobarbital- inducible P-450 LM2 and benzoflavone-inducible P-450 LM4 , is that of Haugen and Coon; it is based on previously described purification procedures carried out in the presence of Renex 690, a nonionic detergent. Highly purified P-450 LM is isolated from 3-methylcholanthrene-treated rats and rabbits and from phenobarbital-treated rats and rabbits by others using different procedures. Liver microsomal cytochrome P-450 (P-450LM) catalyzes several kinds of chemical reactions, including the hydroxylation of fatty acids, steroids, and a variety of foreign compounds, such as drugs and carcinogens. The remarkably broad substrate specificity is partly accounted for by the occurrence of multiple forms of the cytochrome. The purified forms of P- 450 LM are assayed for catalytic activity in substrate hydroxylation in a reconstituted system containing NADPH-cytochrome P-450 reductase and phosphatidylcholine. At all stages of purification, the enzyme preparations are stable for a few weeks at 4° or to storage in the frozen state at -20° for months.

The binding of small hydrocarbon molecules to rabbit liver microsomal cytochrome P-450LM 2 was found to be dependent on several equilibria, including the coordination of the heme sixth ligand and the binding of phospholipid to the protein. There was apparently no substrate binding-associated ionization of the protein in the pH region 6.5 to 9.0. That the binding of substrate with the protein is primarily due to the hydrophobic effect was indicated by correlation of apparent dissociation constants with hydrophobicity, large entropy changes calculated from van't Hoff plots, and the fact that benzphetamine binds only as the unprotonated free amine.

Publisher Summary This chapter describes the reconstitution of the cytochrome P-450-containing mixed-function oxidase system of liver microsomes. Cytochrome P-450 is the predominant enzyme in the membranes of liver cells. It occurs at levels of approximately 10% of the microsomal protein, and the endoplasmic reticulum from which the microsomes are derived represents as much as 90% of the total membranes. This pigment is an unusually versatile catalyst, as shown by its role in hydroxylation and other chemical reactions involving a variety of substrates: drugs, anesthetics, petroleum products, insecticides, carcinogens, and numerous other foreign compounds, as well as naturally occurring substances such as fatty acids and steroids. Liver microsomal cytochrome P-450 (P-450LM) is characterized spectrally as a cytochrome of the b type, but early attempts at solubilization and isolation yielded an altered heme protein, cytochrome P-420, without biological activity.

The transfer of reducing equivalents from NADPH-cytochrome P-450 reductase to P-450LM 4 takes place within a stable complex containing the two enzymes in a 1:1 molar ratio. The reaction proceeds biphasically, with the relative magnitude of the two phases exhibiting a dependence on reduced pyridine nucleotide concentration. Our results appear to indicate that the reductase, rather than the cytochrome, is responsible for the biphasic kinetics observed in this reaction.

By means of second derivative spectroscopy, the resolution of the absorption bands of aromatic amino acids in the middle UV-region in the spectrum of a protein can be improved significantly. This allows more precise assignment of each individual peak to tyrosine, tryptophan, or phenylalanine. With this improved resolution it was shown that in the interaction of the components of the liver microsomal hydroxylation system (P-450LM 2 , NADPH-cytochrome P-450 reductase, and phospholipid) tyrosine residues are involved, participation of phenylalanine residues is likely, and participation of tryptophan residues is excluded.

Publisher Summary (–)t–7, 8–dihydrodiol benzo(a)pyrene (BP) is activated to diol expoxide I and II by the microsomal mixed-function oxidase (MFO) systems. These are major mutagenic and carcinogenic metabolites of BP and bind to DNA, RNA, and proteins. This chapter discusses the role of epoxide hydratase in the hydrolysis and binding of diol epoxides (DE) I and II to DNA and proteins. Studies indicate that DNA diol epoxide binding is stereoselective. In the study discussed in the chapter, it showed that the binding of reactive metabolites formed from 3 H(–)t–7, 8–dihydrodiol BP to DNA and proteins was reduced by epoxide hydratase. The inhibition of DNA and protein binding by epoxide hydratase indicates that the hydratase has some stereospecific interaction with diol epoxides, binds them covalently, alters their mode of hydrolysis, and may reduce their activity as carcinogenic intermediates. Therefore, these results indicate that epoxide hydratase may play a role in diol epoxide detoxification.

The mechanism of the P-450 LM 2 -catalyzed, cumene hydroperoxide-dependent hydroxylation of toluene has been studied by stopped flow spectrophotometry. Addition of cumene hydroperoxide to purified P-450 LM 2 in the absence of toluene caused a pseudo first-order change in the absorbance of the enzyme. Observation of changes at various wavelengths provided the difference and absolute spectra of the intermediate formed. Addition of both cumene hydroperoxide and toluene to P-450 LM 2 produced a type I spectral change in the dead time of the instrument, followed by a pseudo first-order change in the absorbance of the enzyme to give the same spectral intermediate. In these and other studies we have seen no evidence of a classical Compound I of the type seen with peroxidases. In contrast, with P-450 LM 2 the intermediate is formed reversibly and the spectrum varies with the organic moiety of the peroxy compound.

Publisher Summary This chapter describes the purification procedure of fatty acid ω-hydroxylase (alkane hydroxylase) from Pseudomonas oleovorans. The hydroxylation system induced in Pseudomonas oleovorans by growth on alkanes contains three proteins: NADH-rubredoxin reductase—a flavoprotein containing one molecule of flavin adenine dinucleotide (FAD) per polypeptide chain, rubredoxin—a red, nonheme iron protein, and the ω-hydroxylase, which is an unusual example of a mono-oxygenase containing nonheme iron as the prosthetic group. Electron transfer in this system is as follows: NADH reductase → rubredoxin → ω-hydroxylase → O 2 . Ferredoxin-NADP reductase may be substituted for the bacterial reductase, provided that NADPH is used in place of NADH as the primary electron donor. The activity of the hydroxylase is determined as the octane-dependent rate of NADPH oxidation in a reaction mixture containing an excess of reductase and rubredoxin. In the chapter, the various steps are summarized in a table and are carried out at 4°.

Publisher Summary This chapter presents the assay, purification, and properties of β -hydroxy- β -methylglutaryl-coenzyme A (CoA) cleavage enzyme. The metabolism of leucine in animal tissues yields β -hydroxy- β -methylglutaryl-CoA, which undergoes enzymatic aldol cleavage to form equimolar amounts of acetoacetate and acetyl-CoA. The same reaction also occurs during hepatic ketogenesis from fatty acids. The cleavage enzyme has been partially purified from pig heart and beef liver. The acetyl-CoA formed by the action of the cleavage enzyme condenses with oxaloacetate to yield citrate in the presence of the citrate condensing enzyme. Oxaloacetate is formed from L-malate and dinitrophenyl (DPN) by the action of malate dehydrogenase. Thus, the rate of DPN reduction in the presence of an excess of the other assay components is a measure of the amount of cleavage enzyme present. All purification operations are generally carried out at 4°. A highly purified preparation is free of interfering enzymes and therefore, suitable for the studies of the stoichiometry and stereospecificity of the reaction. Enzymatic activity is observed between pH 7.5 and 11.0 with maximal activity at pH 9.5.

Publisher Summary This chapter focuses on the enzymes of isovaleryl CoA metabolism. Isovaleryl CoA, an intermediate in leucine metabolism, is converted to acetoacetate and acetyl CoA by heart and liver enzyme preparations fortified with ATP and bicarbonate. The enzymatic dehydrogenation of isovaleryl CoA and the hydration of the resulting β -methylcrotonyl CoA furnish flhydroxyisovaleryl CoA. 2 The ATP-dependent carboxylation of β -methylcrotonyl CoA is catalyzed by a carboxylase obtained from a mycobacterium or from chicken liver. β -Methylglutaconyl CoA is acted on by a specific hydrase, and the β -hydroxy- β -methylglutaryl CoA formed undergoes enzymatic cleavage to furnish equimolar amounts of acetoaeetate and acetyl CoA. The purification and properties of the carboxylase and the cleavage enzyme from animal tissues are described in the chapter. The chapter also describes the assay method, purification procedure, and properties of β -methylcrotonyl carboxylase from liver and of β -Hydroxy- β -methylglutaryl CoA cleavage enzyme from heart.

Publisher Summary This chapter describes the chemical synthesis of malonic semialdehyde, β‑hydroxypropionate, and β‑hydroxyisobutyrate. For synthesis of malonic semialdehyde, ethyl β,β-diethoxypropionate, made by the procedure of Sorm and Smrt is converted to malonic semialdehyde. The ester acetal is obtained in considerably higher yield by this procedure than by the conversion of ethyl formate and ethyl acetate to the sodium derivative of malonic semialdehyde ethyl ester, and subsequent treatment with hydrogen chloride in absolute ethanol. β-Hydroxypropionic acid preparations obtained commercially or made from ethylene cyanohydrin contain varying amounts of impurities—chiefly a material which may be a polyester acid of the type described by Gresham. Concentrated solutions of β-hydroxypropionic acid develop these impurities on storage. β-hydroxypropionic acid generated from propiolactone is purified as the crystalline calcium–zinc salt. Solutions of potassium β‑hydroxypropionate are then generated as needed by treating the calcium–zinc salt with Dowex 50 (K + form). The dl -β-hydroxyisobutyric acid is prepared from formaldehyde and ethyl α-bromopropionate by Reformatsky reaction essentially according to the procedure of Blaise and Herman.

Publisher Summary The treatment of a solution of biotin and adenylic acid in pyridine with dicyclohexylcarbodiimide leads to the formation of biotinyl 5′-adenylate. The crude product is purified by chromatography on diethylaminoethyl (DEAE) cellulose. The preparation and purification of this compound are described in this chapter. Biotinyl 5′-adenylate is an intermediate in the enzymatic conversion of d -biotin to biotinyl-CoA and has been identified as an intermediate in the conversion of propionyl-coenzyme A (CoA) apocarboxylase to the biotin-containing holocarboxylase, catalyzed by a synthetase from liver. Biotinyl adenylate is similarly involved in the synthesis of holotranscarboxylase, β -methylcrotonyl-CoA holocarboxylase, and apparently carboxylase.