Dieter Müller-Enoch

Inhibition of Cytochrome P450 Mediated Enzyme Activity by Alkylphosphocholines

Abstract The inhibitory potency of four alkylphospholipids: rac-1-O-phosphocholine-2-hydroxy-octadecane (rac-2-OH), rac-1-O-phosphocholine-2-O-acetyl-octadecane (rac-2-O-acetyl), rac-1- O-phosphocholine-2-amino-octadecane (rac-2-NH2) and rac-1-O-phosphocholine-2-N-acetyloctadecane (rac-2-N-acetyl), on the cytochrome P450-dependent monooxygenase activity has been evaluated. The IC50 values of the alkylphosphocholines with 7-ethoxycoumarin as substrate in liver microsomal fractions of PB-treated rats and with a reconstituted CYP2B1: NADPH-P450-reductase system are in the range of 3.2D5.0 μм and 2.8D3.5 μм, respectively. Lineweaver-Burk plots with the inhibitors in concentrations that were found to cause roughly a 50% inhibition and with 7-ethoxycoumarin as substrate revealed for all four alkylphospholipids a competitive inhibition type. The degree of the competitive inhibition is quantified by the Ki values. With liver microsomal fractions of PB-treated rats, the Ki values of rac-2-OH (Ki = 1.36 μм) and rac-2-O-acetyl (Ki = 1.33 μm) differs slightly from those of rac-2-NH2 (Ki = 2.2 μм) and rac-2-N-acetyl (Ki = 2.2 μм), but with the reconstituted CYP2B1: NADPHP450- reductase system all Ki values are in the small range of 1.8 D 2.6 μм, indicating that the short substituted group at the 2-position (OH; O-acetyl; NH2; N-acetyl) of the long chain octadecanol part of the phosphodiesters exhibit no essential role on the strong inhibitory potency of these alkylphosphocholines on the 7-ethoxycoumarin-O-deethylase activity.

Regioselective O-demethylation of scoparone: differentiation between rat liver cytochrome P-450 isozymes

Abstract The ratios of the scoparone O-demethylation products scopoletin to isoscopoletin were deter mined for reconstituted complexes of NADPH-P-450 reductase and each of four P-450 isozymes in a 2:1 molar ratio with a 1:1 mixture of [7-O-methyl-14C]-and [6-O-methyl-14C]-scoparone as substrate. The two phenobarbital inducible forms P-450PB-B and P-450PB-D have a 1:0.8 ± 0.05 scopoletin to isoscopoletin ratio, and the two β-naphthoflavone inducible forms P-450βNF-B and P-450βNF/ISF-G have ratios of 1:4.4 ± 0.1 and 1:3.8 ± 0.1, respectively. The scoparone-O-demethylation activities of the reconstituted preformed complexes of the four P-450 isozymes are given.

Enzyme Activity of the Cytochrome P-450 Monooxygenase System in the Presence of Single Chain Lipid Molecules

Abstract The influence of single chain lipids on the 7-ethoxycoumarin O-deethyase activity of the reconstituted binary protein complex of isolated cytochrome P450 and NADPH-cytochrome P450 reductase has been examined. The enzyme activity of this binary enzyme complex has been shown to be influenced by (i) altering the complexation process of both proteins, (ii) by altering the catalytic cycle time of the active binary protein complex and (iii) by altering the fraction of substrate molecules at the catalytic center of the enzyme. Competitive inhibition was measured for all single chain molecules. The following dissociation coefficients of sub strate and lipids used for the catalytic center of the protein were obtained: 110 μм 7-ethoxy-coumarin (substrate), 1.1 μм MOG (1-monooleoyl-rac-glycerol), 0.3 μм SPH (D-sphingosine), 1.5 μм OA (oleic acid), 3.0 μм LPC (L-a-lysophosphatidyl-choline), 15.5 μм MSG (1-mono-stearoyl-rac-glycerol), 9.5 μм AA (arachidonic acid), 9.0 μм PaCar (palmitoyl-L-carnitine), 3.5 μм MPG (2-monopalmitoyl-glycerol), 1.5 μм LPI (L-a-lysophosphatidyl-inositol), 50 μм LA (lauric acid), 60 μм MA (myristic acid). 85 μм PA (palmitic acid), >100 μм SA (stearic acid). Only competitive inhibition with the substrate molecule 7-ethoxycoumarin was observed for the single chain lipids LA, MA, PA, SPH, SA, and OA. Non-competitive effects were observed for MPG (-0.03 μм-1) , PaCar (-0.02 μм-1) , MSG (-0.023 μм-1) , LPC (-0.03 μм-1) , A A (-0.03 μм-1) , and MOG (+0.04 μм-1). The negative sign indicates that the cycle time of the working binary complex is enlarged. The positive sign indicates that the formation of the binary complex is enhanced by MOG.

Complexation of membrane-bound enzyme systems.

Abstract The effect of changes in the N-terminal membrane-binding domain of cytochrome P450 forms and NADPH-cytochrome P450 reductase types on the cytochrome P450-dependent monooxygenase activities, has been examined. The nifedipine oxidase activity of two human P450 forms (CYP3A4, CYP3A4NF14) which differ only in their primary structure by ten amino acid residues in the N-terminal membrane-binding domain, yields nearly the same catalytic cycle time τ =2.65 ± 0.15 s, due to their identical cytosolic catalytic protein structure. In contrast, the complex formation process ([P450]+[reductase]↔[complex]) described by the dissociation constant KD at high substrate concentration ([S]>>KS) and low product concentration ( [ P ]<<Kp ) is determined to be KD/[P450]ᴏ = 0.3 and 2.0, respectively. These values reflect large differences in the affinity of both P450 forms for the same type of reductase which is only due to their modified membrane-binding domains. In the present work, it has been shown for the first time, that the membrane-binding domain of cytochrome P450 enzymes determines the complexation process of the binary P450:reductase system. Furthermore, the nifedipine oxidase activity of the human CYP3A4 form reconstituted with two different types of reductase from human and rabbit also has the same catalytic cycle time τ = 2.65 ± 0.15 s. This result is based on the similarity of the primary structure of the cytosolic catalytic domain of both reductase types. However, the complex was formed with different dissociation constants of KD/[P450]ᴏ = 0.3 and 4.7, respectively. This different affinity of both reductase types to the same P450 form is interpreted as a consequence of the substantial alteration of the amino acids in the N-terminal primary structure of their membrane-binding domains. 7-Ethoxycoumarin O-deethylase activity of two rat P450 forms (CYP2B1 and CYP1A1) were reconstituted with the same rat reductase. The catalytic cycle time for each P450 form is τ = 1.8 and 0.6 s, respectively. Correspondingly, the complex formation process controlled by the dissociation constant KD has changed from KD/[P450]ᴏ = 2.3 to 1.7, respectively. This is because both forms differ in their cytosolic as well as in their membranebinding domains.

Blue light mediated photoreduction of the flavoprotein NADPH-cytochrome P450 reductase. A Förster-type energy transfer.

The absorption spectra and the corresponding molar absorption coefficients of the fluorophores umbelliferone, FAD and FMN and of the FAD and FMN containing flavoprotein NADPH-cytochrome P450 reductase of different oxydation-reduction states are documented. Binding spectra of the ligand umbelliferone with the CYP2B1: NADPH-cytochrome P450 reductase-complex were determined by difference spectroscopy. The Scatchard plot of the equilibrium ligand binding shows a high affinity part and a low affinity part of 12 and 34 umbelliferone binding sites per CYP2B1: reductase-complex molecule, respectively The fluorescence excitation and emission spectra of the donor molecule umbelliferone and the acceptor molecules FAD and FMN are given. The fluorescence spectra of the reaction components under test conditions of CYP2B1-dependent 7-ethoxycoumarin-O-deethylase are measured. The excitation energy transfer from the donor umbelliferone (λE=380 nm; λF= 460 nm) to the acceptor molecule FMN (λE=465 nm; λF=525 nm) was examined under assay conditions. The results dem onstrate that a radiationless Förster-type energy transfer takes place in the presence of the CYP2B1: reductase-complex. It turned out that this effect is a function of the protein complex-concentration. The data presented here combined with previously made observations by Müller-Enoch (Müller-Enoch D. (1994), Z. Naturforsch. 49c, 763-771) support the finding that the umbelliferone molecules, n = 12-34, bound per mole of CYP2B1: reductase-complex, transfer their absorbed light energy radiationless to the FAD binding domain. The complex formed containing 12 or 34 molecules of umbelliferone provides absorption coefficient values at λ= 380 nᴍ of 78 and 221 mᴍ-1 · cᴍ-1 , respectively. The Förster-type energy transfer from the donor umbelliferone to the acceptor FAD not only leads to a light activation of the singlet state of FAD but also to a conformational change of the amino acids close to the FAD binding side to favour the encaging of the FAD* triplet state which reacts with the NADPH to form the FADH2 reductase. Due to this process the overall reaction can start with the unquenched excited FAD* triplet state as an interm ediate which is about 30 kJ/mol lower in energy than the dark reaction