Reconstitution Premixes for Assays Using Purified Recombinant Human Cytochrome P450, NADPH-Cytochrome P450 Reductase, and Cytochromeb5

doi:10.1006/abbi.1997.0378

Archives of Biochemistry and Biophysics

Volume 348, Issue 1, 1 December 1997, Pages 107-115

https://doi.org/10.1006/abbi.1997.0378 Get rights and content

Abstract

The development of enzyme and buffer premixes forin vitrobiotransformation assays is described. The protein premixes contain a mixture of three recombinant human proteins, cytochrome P450 (P450) 3A4, NADPH-P450 reductase, cytochromeb₅, and liposomes. The buffer premix contains reagents which, when diluted, provide for optimal metabolic activity with selected P450 3A4 substrates. P450 3A4 premixes were competent in the oxidation of known substrates including testosterone, midazolam, nifedipine, erythromycin, benzphetamine, and amitriptyline. Premixes stored at −80°C for 2 months and those that underwent an additional five freeze/thaw cycles were able to hydroxylate testosterone at turnover rates similar to freshly prepared reconstitution mixes. In addition, premixes stored unfrozen at 4°C for 2 weeks showed no significant loss in the rate of testosterone 6β-hydroxylation by P450 3A4. Premixes prepared with and without reduced glutathione, a component which had previously been found to be important for P450 3A4 reactions, were equally efficient at carrying out testosterone hydroxylation under these conditions. Kinetic parameters determined for the metabolism of testosterone, amitriptyline, nifedipine, and benzphetamine using P450 3A4 premixes were compared with human pooled microsomes and insect microsomes prepared from cells infected with a baculovirus containing two cDNA inserts coding for P450 3A4 and NADPH-P450 reductase. Each format gave differentV_maxandK_mvalues indicating different catalytic efficiencies. Analysis of P450 1A2 premixes which contained different lipid concentrations indicated thatV_maxandK_mcould be altered. The availability of human P450 recombinant enzymes and the development of the P450 premixes that remain active after being stored frozen should allow for rapid identification of novel P450 substrates and inhibitors and the development of large-scale screening assays.

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Cytochrome P450 (P450) 3A4 is the enzyme most involved in the metabolism of drugs and can also oxidize numerous steroids. This enzyme is also involved in one-half of pharmacokinetic drug–drug interactions, but details of the exact mechanisms of P450 3A4 inhibition are still unclear in many cases. Ketoconazole, clotrimazole, ritonavir, indinavir, and itraconazole are strong inhibitors; analysis of the kinetics of reversal of inhibition with the model substrate 7-benzoyl quinoline showed lag phases in several cases, consistent with multiple structures of P450 3A4 inhibitor complexes. Lags in the onset of inhibition were observed when inhibitors were added to P450 3A4 in 7-benzoyl quinoline O-debenzylation reactions, and similar patterns were observed for inhibition of testosterone 6β-hydroxylation by ritonavir and indinavir. Upon mixing with inhibitors, P450 3A4 showed rapid binding as judged by a spectral shift with at least partial high-spin iron character, followed by a slower conversion to a low-spin iron–nitrogen complex. The changes were best described by two intermediate complexes, one being a partial high-spin form and the second another intermediate, with half-lives of seconds. The kinetics could be modeled in a system involving initial loose binding of inhibitor, followed by a slow step leading to a tighter complex on a multisecond time scale. Although some more complex possibilities cannot be dismissed, these results describe a system in which conformationally distinct forms of P450 3A4 bind inhibitors rapidly and two distinct P450–inhibitor complexes exist en route to the final enzyme–inhibitor complex with full inhibitory activity.
Expression of active human P450 3A4 on the cell surface of Escherichia coli by Autodisplay
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The cytochrome P450 enzyme system comprises a large group of enzymes catalyzing a broad diversity of reactions and an extensive substrate specificity, which makes them the most versatile known catalysts. CYP3A4 is one of the important human P450 enzymes and involved in the oxidation of a large range of substrates including toxins and pharmaceuticals. Bottlenecks in studying this enzyme include the difficulty in expressing it in a bacterial host, its need for membrane surroundings and the limited substrate accessibility of enzymes expressed within the cell. To circumvent these difficulties, human CYP3A4 was expressed on the outer membrane of Escherichia coli using Autodisplay. Transport of CYP3A4 to the cell surface was monitored by SDS-PAGE and Western blot analysis of outer membrane proteins. Localization on the cell envelope was determined by flow cytometry after immunolabeling, a whole cell ELISA and a protease accessibility assay. A HPLC assay confirmed the catalytic activity of displayed CYP3A4, using testosterone as a substrate. This activity required the external addition of electron supplying enzymes, however surprisingly, we found that the external addition of a heme group was not necessary. Our results indicate that human CYP3A4 can be recombinantly expressed by surface display in a gram-negative bacterium.
Cooperativity in monomeric enzymes with single ligand-binding sites
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Cooperativity is widespread in biology. It empowers a variety of regulatory mechanisms and impacts both the kinetic and thermodynamic properties of macromolecular systems. Traditionally, cooperativity is viewed as requiring the participation of multiple, spatially distinct binding sites that communicate via ligand-induced structural rearrangements; however, cooperativity requires neither multiple ligand binding events nor multimeric assemblies. An underappreciated manifestation of cooperativity has been observed in the non-Michaelis–Menten kinetic response of certain monomeric enzymes that possess only a single ligand-binding site. In this review, we present an overview of kinetic cooperativity in monomeric enzymes. We discuss the primary mechanisms postulated to give rise to monomeric cooperativity and highlight modern experimental methods that could offer new insights into the nature of this phenomenon. We conclude with an updated list of single subunit enzymes that are suspected of displaying cooperativity, and a discussion of the biological significance of this unique kinetic response.
Conversion of 7-dehydrocholesterol to 7-ketocholesterol is catalyzed by human cytochrome P450 7A1 and occurs by direct oxidation without an epoxide intermediate
2011, Journal of Biological Chemistry
Citation Excerpt :
Enzyme reaction mixtures for P450 7A1 reactions typically contained 0.2 μm P450 7A1, 3.0 μm NADPH:P450 reductase, 60 μm l-α-1,2-dilauroyl-sn-glycero-3-phosphocholine, 7-dehydrocholesterol in 3.1 mm HPβCD (0.45%, w/v), and 0.41 mm Tween 20 (0.05%, v/v) in 50 mm potassium phosphate buffer (pH 7.4) (40). For P450 3A4 reactions, a “5× P450 protein premix” was prepared with slight modification of a previously described method (45). Briefly, a 5× protein premix including 2.5 μm P450 3A4, 5 μm NADPH:P450 reductase, 2.5 μm cytochrome b5, 150 μg/ml phospholipid mixture (1:1:1 (w/w/w) l-α-1,2-dioleoyl-sn-glycero-3-phosphocholine/l-α-1,2-dilauroyl-sn-glycero-3-phosphoserine/l-α-1,2-dilauroyl-sn-glycero-3-phosphocholine), 0.50 mg/ml potassium CHAPS (0.77 mm), and 3.0 mm GSH in 50 mm potassium HEPES buffer (pH 7.4) and a 5× buffer mix including 12 mm GSH and 150 mm MgCl2 in 200 mm potassium HEPES buffer (pH 7.4) were prepared.
7-Ketocholesterol is a bioactive sterol, a potent competitive inhibitor of cytochrome P450 7A1, and toxic in liver cells. Multiple origins of this compound have been identified, with cholesterol being the presumed precursor. Although routes for formation of the 7-keto compound from cholesterol have been established, we found that 7-dehydrocholesterol (the immediate precursor of cholesterol) is oxidized by P450 7A1 to 7-ketocholesterol (k_cat/K_m = 3 × 10⁴ m⁻¹ s⁻¹). P450 7A1 converted lathosterol (Δ⁵-dihydro-7-dehydrocholesterol) to a mixture of the 7-keto and 7α,8α-epoxide products (∼1:2 ratio), with the epoxide not rearranging to the ketone. The oxidation of 7-dehydrocholesterol occured with predominant formation of 7-ketocholesterol and with the 7α,8α-epoxide as only a minor product; the synthesized epoxide was stable in the presence of P450 7A1. The mechanism of 7-dehydrocholesterol oxidation to 7-ketocholesterol is proposed to involve a Fe^III-O-C-C⁺ intermediate and a 7,8-hydride shift or an alternative closing to yield the epoxide (Liebler, D. C., and Guengerich, F. P. (1983) Biochemistry 22, 5482–5489). Accordingly, reaction of P450 7A1 with 7-[²H₁]dehydrocholesterol yielded complete migration of deuterium in the product 7-ketocholesterol. The finding that 7-dehydrocholesterol is a precursor of 7-ketocholesterol has relevance to an inborn error of metabolism known as Smith-Lemli-Opitz syndrome (SLOS) caused by defective cholesterol biosynthesis. Mutations within the gene encoding 7-dehydrocholesterol reductase, the last enzyme in the pathway, lead to the accumulation of 7-dehydrocholesterol in tissues and fluids of SLOS patients. Our findings suggest that 7-ketocholesterol levels may also be elevated in SLOS tissue and fluids as a result of P450 7A1 oxidation of 7-dehydrocholesterol.

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Regular ArticleReconstitution Premixes for Assays Using Purified Recombinant Human Cytochrome P450, NADPH-Cytochrome P450 Reductase, and Cytochromeb5☆

Abstract

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Biochem. Biophys. Acta

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

Regular Article
Reconstitution Premixes for Assays Using Purified Recombinant Human Cytochrome P450, NADPH-Cytochrome P450 Reductase, and Cytochromeb₅☆