Cytochrome P‐450: hexameric structure of the purified LM4 form

Myasoedova, K. N.; Tsuprun, Vladimir

doi:10.1016/0014-5793(93)81083-c

Cited by 23 publications

(21 citation statements)

References 22 publications

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“…Analytical ultracentrifugation studies with CYP2C9 and other proteins have shown that these P450 proteins exist in a highly heterogeneous environment, forming up to 20mers (M. Subramanian and T. S. Tracy, unpublished data;Guengerich and Holladay, 1979;French et al, 1980;Tsuprun et al, 1986;Myasoedova and Tsuprun, 1993;Black and Martin, 1994;Kempf et al, 1995;Davydov et al, 2005). Chemical cross-linking and bimolecular fluorescence complementation spectroscopy studies have shown the formation of CYP1A1-CYP3A2 and CYP2E1-CYP2C2 complex formation, respectively (Alston et al, 1991;Davydov et al, 2005;Ozalp et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

CYP2C9-CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N Terminus

Subramanian

Tam²,

Zheng³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Cytochromes P450 (P450s) interact with redox transfer proteins, including P450 reductase (CPR) and cytochrome b 5 (b5), all being membrane-bound. In multiple in vitro systems, P450-P450 interactions also have been observed, resulting in alterations in enzymatic activity. The current work investigated the effects and mechanisms of interaction between CYP2C9 and CYP3A4 in a reconstituted system. CYP2C9-mediated metabolism of S-naproxen and S-flurbiprofen was inhibited up to 80% by coincubation with CYP3A4, although K m values were unchanged. Increasing CYP3A4 concentrations increased the degree of inhibition, whereas increasing CPR concentrations resulted in less inhibition. Addition of b5 only marginally affected the magnitude of inhibition. In contrast, CYP2C9 did not alter the CYP3A4-mediated metabolism of testosterone. The potential role of the hydrophobic N terminus on these interactions was assessed by incubating truncated CYP2C9 with full-length CYP3A4, and vice versa. In both cases, the inhibition was fully abolished, indicating an important role for hydrophobic forces in CYP2C9-CYP3A4 interactions. Finally, a CYP2C9/CYP3A4 heteromer complex was isolated by coimmunoprecipitation techniques, confirming the physical interaction of the proteins. These results show that the N-terminal membrane binding domains of CYP2C9 and CYP3A4 are involved in heteromer complex formation and that at least one consequence is a reduction in CYP2C9 activity.Cytochromes P450 (P450s) are the primary oxidative enzymes responsible for the metabolism of xenobiotics by humans and animals (Guengerich, 2006). P450s can oxidize structurally diverse substrates ranging from small hydrocarbons to large molecules such as cyclosporine. P450s function by transferring electrons via a P450 catalytic pathway, wherein the first electron is transferred to the P450 via cytochrome NADPH P450 reductase (CPR) and the second electron is transferred via either CPR or cytochrome b 5 (b5) (Guengerich, 2002). Inefficiencies in the catalytic pathway, in part, govern the rate of transformation. P450s and CPR are bound to the cell membrane via a hydrophobic N terminus, whereas b5 is bound via its C terminus, each terminal being composed of 30 to 60 amino acids (Szczesna-Skorupa and Kemper, 2008). This hydrophobic region also contributes to protein aggregation, resulting in the formation of high molecular weight polymers in membranes and reconstituted systems (SzczesnaSkorupa and Kemper, 2008). Considering that P450s and CPR, and P450s and b5 interact within the cell membrane, P450-P450 interactions also appear conceivable. Indeed, in vitro evidence of P450-P450 interactions have been observed in several studies, including interactions of human CYP2C9-CYP2C19, rabbit CYP1A2-CYP2B4, rabbit CYP1A2-CYP2E1, human CYP3A4-CYP1A1, human CYP3A4-CYP1A2, and human CYP2C9-CYP2D6 (Cawley et al., 1995;Yamazaki et al., 1997;Backes et al., 1998;Backes and Cawley, 1999;Kelley et al., 2005Kelley et al., , 2006Subramanian et al., 2009). Substrate-dependent activation o...

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Section: Discussionmentioning

confidence: 99%

CYP2C9-CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N Terminus

Subramanian

Tam²,

Zheng³

et al. 2010

Drug Metab Dispos

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“…The dimers referred to are the side-to-proximal dimers described above. The arrangement is intriguing in that it is consistent with functional membrane binding and has similar features to the hexameric structures of CYP1A2 (Myasoedova and Tsuprun, 1993) and CYP2B4 (Tsuprun et al, 1986) observed by electron microscopy as it is comprised of two types of contacts between the CYP2C9 units of the hexamer. In addition to the side-toproximal interaction, the three sets of dimers are joined together with threefold symmetry along the distal faces and the side between the proximal and distal faces that contains the regions between the B/C helices and the H/I helices in addition to the C-terminus of the enzyme.…”

Section: Putative Hexameric Quaternary Structure Of Membrane-bound Cymentioning

confidence: 67%

“…Determination of P450-P450 Binding Affinities Purified P450s have a remarkable tendency to aggregate in aqueous solutions (Guengerich and Holladay, 1979;French et al, 1980;Dean and Gray, 1982;Myasoedova and Berndt, 1990;Myasoedova and Tsuprun, 1993). The arrangement of purified, detergent-free homomeric CYP1A2 (as well as CYP2B4) in solution was defined by electron microscopy as being hexameric with two layers of P450 trimers that are comprised of two types of distinct P450-P450 interactions (Tsuprun et al, 1986;Myasoedova and Tsuprun, 1993).…”

Section: Physical Studies Of Membrane Systems Containing P450smentioning

confidence: 99%

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

Pandey¹,

Henderson²,

Ishii³

et al. 2018

Frontiers Research Topics

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“…Electrons required for monooxygenase reactions are mainly supplied by membrane-associated flavoprotein, NADPH-cytochrome P450 reductase. Purified cytochromes P450 and the reductase were shown to form oligomers (French et al 1980;Tsuprun et al 1986;Myasoedova 1998;Myasoedova and Tsuprun 1993). However, there is no generally accepted conception concerning organization of the two proteins within the membrane and their mechanism of interaction, as well as the minimal size of the functional unit.…”

Section: Introductionmentioning

confidence: 95%

“…However, there is no generally accepted conception concerning organization of the two proteins within the membrane and their mechanism of interaction, as well as the minimal size of the functional unit. Our previous data suggest the existence of universal hexameric organization of hepatic cytochromes P450 (Myasoedova and Tsuprun 1993;Myasoedova and Magretova 2001;Myasoedova and Stel'mashchuk 2003).…”

Section: Introductionmentioning

confidence: 96%

Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

2006

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Mechanism of substitution of nonionic detergent Emulgen 913 for phospholipid as an activator of N-demethylase activity of cytochrome P450 form 2B4 (LM(2)) has been studied. It is shown that such an activation takes place at the detergent concentrations below values critical for micelle formation. Under these conditions, Emulgen does not affect the hexameric state of the cytochrome. The stimulating effect proved to be similar in reconstituted monooxygenase systems containing (a) cytochrome P450 2B4 and NADPH-cytochrome P-450 reductase and (b) cytochrome 2B4 and organic hydroperoxides. These results indicate that the activation is due to an effect of the detergent upon P450 2B4 per se rather than upon P450/flavoprotein complex formation. The above conclusion is supported by the sedimentation data and measurement of the CD spectra of cytochrome P450 2B4 at 380-450 nm.

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Cytochrome P‐450: hexameric structure of the purified LM4 form

Cited by 23 publications

References 22 publications

CYP2C9-CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N Terminus

CYP2C9-CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N Terminus

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

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