Typologie

Backes, Uwe

doi:10.1007/978-3-322-86110-8_5

Cited by 8 publications

(8 citation statements)

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“…The biphasic rate constants are 4.1 and 0.51 s Ϫ1 , and the amplitude of the fast phase, k 1 , is 81% (see Table 1). This result is similar to that reported by other investigators (29,30). In the presence of holocyt b 5 , the rate of reduction of ferric cyt P450 2B4 decreases as observed for cyt P450 1A2 and 2E1 (31).…”

Section: Kinetics Of Reduction Of Ferric Cyt P450 2b4 By Cpr In the Psupporting

confidence: 92%

Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4

Zhang

Hamdane²,

Im³

et al. 2008

Journal of Biological Chemistry

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Microsomal cytochromes (cyt) 3 P450, functioning as monooxygenases, catalyze the oxidative biotransformation of numerous pharmaceuticals, carcinogens, pro-carcinogens, and endogenous compounds like fatty acids and steroids. Cyts P450 require two electrons and two protons to carry out catalysis that leads to insertion of a single oxygen atom into the substrate. In the mammalian microsomal cyt P450 system, the two electrons are delivered to cyt P450 by NADPH-dependent cytochrome P450 reductase (CPR). Like cyt P450, CPR is membrane-bound and located in the membrane of the endoplasmic reticulum. CPR contains two flavin molecules, FMN and FAD. The diflavin moiety of CPR is essential for sequential electron transfer to cyt P450 as it permits CPR to accept two electrons from NADPH and transfer one electron at a time to cyt P450. The first electron from CPR reduces ferric cyt P450 to ferrous cyt P450, which rapidly binds oxygen to form oxyferrous cyt P450. The second electron is then delivered to oxyferrous cyt P450. This is followed by protonation of the reduced oxyferrous intermediate leading to heterolytic cleavage of the oxygen bond to form water and an oxyferryl intermediate, the putative, active, oxidizing species of cyt P450. An oxygen atom is inserted into the substrate, and the more hydrophilic product dissociates from the enzyme. Readers are referred to a recent review for further details about the cyt P450 reaction cycle (1).An alternative electron donor to cyt P450 is cyt b 5 , another microsomal hemoprotein also located in the endoplasmic reticulum membrane. Because of its relatively high mid-point redox potential (ϩ25 mV versus NHE), cyt b 5 can deliver only the second electron to oxyferrous cyt P450 but not the first electron to ferric cyt P450. It has been recognized for 3 decades that cyt b 5 may either increase, decrease, or not alter the activity of selected cyts P450 (2, 3). Cyt b 5 has been reported to affect the catalytic activity of more than 20 cyt P450 isoforms, including the majority of the human drug-metabolizing cyt P450 isoforms like cyt P450 3A4, 2B6, 2C9, 2C19, and 2E1 (1, 4 -8). The effect of cyt b 5 has also been shown to depend on the cyt P450 isozyme and substrate (2, 9). In the case of cyt P450 2B4 and 2E1, the electron donating properties of cyt b 5 are required for its stimulatory activity (6, 7, 9 -11), although some studies suggest that apo-cyt b 5 can stimulate the activity of cyt P450 3A4 via an allosteric effect (12). At present, the ability of apo-cyt b 5 to stimulate cyt P450 3A4 is controversial (13). Experiments performed in the reconstituted system with purified proteins have demonstrated that ferrous cyt b 5 can rapidly reduce oxyferrous cyt P450 2B4 (14,15).It is known that cyt P450 2B4 forms a 1:1 complex with CPR and with cyt b 5 in a purified, reconstituted system (16,17). A site-directed mutagenesis study of the interactions of cyt P450 2B4 with CPR and cyt b 5 has identified residues, primarily in the * This work was supported by National Institutes of Health Grant ...

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Section: Kinetics Of Reduction Of Ferric Cyt P450 2b4 By Cpr In the Psupporting

confidence: 92%

Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4

Zhang

Hamdane²,

Im³

et al. 2008

Journal of Biological Chemistry

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“…The first accounts of the reduction kinetics of P450s described biphasic kinetics (58,59). Successive reports also present biphasic kinetics even with purified P450 s (24, 25, 40 -42, 49, 52, 60), although one of these reports indicates that 90% of the reduction was done in the fast phase (60). Most of these studies were done at 25°C or less, and part of the reason we saw primarily single exponential reduction kinetics may be due to the 37°C temperature we used (for comparison to steady-state results).…”

Section: Discussionmentioning

confidence: 88%

Lack of Electron Transfer from Cytochrome b5 in Stimulation of Catalytic Activities of Cytochrome P450 3A4

Yamazaki

Johnson

Ueng

et al. 1996

Journal of Biological Chemistry

179

116

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Many catalytic activities of cytochrome P450 (P450)3A4 More than 40 P4501 enzymes are found in a single mammalian species (2). The proteins constitute a superfamily and collectively contribute extensively to the oxidation of xenobiotic chemicals (e.g. drugs, carcinogens, pesticides, alkaloids, and other natural products) and also endobiotics (e.g. steroids, eicosanoids, fat-soluble vitamins, fatty acids) (3-6). The contributions of these P450 enzymes to metabolism in humans are well recognized, particularly regarding issues of drug clearance (7-9). There is general agreement that, in most humans, P450 3A4 is the most abundant of the P450s in both liver and small intestine (8, 9); it can constitute up to 60% of the total P450 in the liver (10). The intestinal enzyme has been implicated in variation in the bioavailability of many orally administered drugs (11). P450 3A4 has a very broad range of substrates, with more than 60 drugs having been already identified (9). These vary widely in structure, and one of the questions about this enzyme has been the molecular basis of its broad catalytic specificity (12, 13). Other mechanistic questions involve the basis of the sigmoidal plots of enzyme velocity versus substrate seen with some compounds (14 -16) and the stimulation of activity by chemicals other than the substrate (14,17,18). The purified enzyme, along with other P450 3A subfamily enzymes, is much more sensitive to its reconstitution environment than are most other P450s (19, 20). A variety of components have been reported to stimulate catalytic activity, including long chain unsaturated phosphatidylcholines (21), phosphatidylserine (20,22), ionic detergents (21, 22), GSH (23), divalent cations (24, 25), and b 5 (19,21). Not all of these components are directly relevant to the membrane-bound enzyme, but Mg 2ϩ has been shown to stimulate activity of the enzyme in microsomes (24, 25) and antibodies raised against b 5 can inhibit some catalytic activities of P450 3A4 in microsomes (19,25). Somewhat surprisingly, certain catalytic activities of P450 3A4 are quite refractory to alterations in lipids and b 5 (24, 26).In order to better understand this complex but important system, we initiated a systematic investigation of some of the system components on individual steps in the catalytic cycle of purified recombinant P450 3A4 (14,19,20,(23)(24)(25). A general conclusion about the role of b 5 in modulating P450 reactions has been that electron transfer from b 5 to P450 occurs in step 4 of Scheme 1 (27). Recently we found qualitative evidence that b 5 could also stimulate the reduction of P450 3A4 by the flavoprotein NADPH-P450 reductase (24). We now report that apo-b 5 (devoid of heme) can replace b 5 in the efficient oxidation of the prototypic P450 substrates testosterone and nifedipine and that apo-b 5 can also replace b 5 in the facilitating electron flow from NADPH-P450 reductase to P450 3A4, in the absence of electron transfer from b 5 or modulation of the E m,7 of P450 3A4.

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“…NADPH-cytochrome P450 reductase was purified from phenobarbital-treated rabbits as described previously (10). Recombinant rabbit NADPH cytochrome P450 reductase (plasmid: pSC-CPR, provided by Dr. Lucy Waskell (Univ.…”

Section: Enzymesmentioning

confidence: 99%

Inhibition of CYP2B4 by the mechanism-based inhibitor 2-ethynylnaphthalene: Inhibitory potential of 2EN is dependent on the size of the substrate

Cheng

Reed

Harris

et al. 2007

Archives of Biochemistry and Biophysics

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Abstract2-Ethynylnaphthalene (2EN) is a mechanism-based inhibitor of CYP2B4 with two components to the inhibition, (1) enzyme inactivation, which requires covalent binding of the 2EN metabolite, and (2) reversible inhibition by 2EN itself. Both inhibitory components were examined using several different CYP2B4 substrates. Preincubation of CYP2B4 with 2EN led to a time-dependent inactivation of each of the CYP2B4-dependent activities examined; however, the ability of 2EN to reversibly inhibit CYP2B4 depended on the substrate employed, which is inconsistent with classical inhibition patterns. The degree 2EN's reversible inhibition was shown not to correlate with the substrate affinity for the active site, but with parameters related to the molecular size of the substrate. The results are consistent with 2EN and the smaller substrates simultaneously fitting in the CYP2B4 active site, leading to very little inhibition. Larger substrates exhibited greater degrees of inhibition because of their inability to co-bind with inhibitor in the active site.Cytochrome P450 enzymes play a major role in oxidizing an extensive array of compounds including drugs, pesticides, carcinogens, steroids and fatty acids. The identification of specific inhibitors and substrates for several of the P450 families has aided in the characterization of relative enzyme levels in microsomal preparations and has been useful as probes of the structure of the active site of specific P450 enzymes. Cytochrome P450 inhibitors can be divided into three categories that differ in mechanism: (a) agents that bind reversibly; (b) agents that form quasi-irreversible complexes with the heme iron atom, and (c) agents that bind irreversibly to the protein or the prosthetic heme group, or that accelerate degradation of the prosthetic heme group (1). The second and third agents fall into the category of mechanism-based (or suicide) inhibitors. Three mechanisms for the inactivation of P450 by the mechanism-based inhibitors are known: N-alkylation of the prosthetic heme group; destruction of the heme; and covalent modification of the apoprotein by the reactive intermediate. The mechanism-based inactivation of P450 enzymes involves metabolic activation by the enzyme itself and is characterized by a time-dependent decrease in enzymatic activities. Dilution or dialysis of the enzyme:inhibitor solution does not dissociate the EI complex and restore enzyme activity. Reversible inhibitors interact with the enzyme through noncovalent association/dissociation reactions, and do not require prior metabolism of the inhibitor.Corresponding author: Wayne L. Backes, Ph.D., Department of Pharmacology & The Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar St., New Orleans, LA 70112, wbacke@lsuhsc.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typese...

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Typologie

Cited by 8 publications

References 0 publications

Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4

Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4

Lack of Electron Transfer from Cytochrome b5 in Stimulation of Catalytic Activities of Cytochrome P450 3A4

Inhibition of CYP2B4 by the mechanism-based inhibitor 2-ethynylnaphthalene: Inhibitory potential of 2EN is dependent on the size of the substrate

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