Conformational Change of Cytochrome P450 1A2 Induced by Sodium Chloride

Yun, Chul‐Ho; Song, Myeonghun; Ahn, Taeho; Kim, Hyoungman

doi:10.1074/jbc.271.49.31312

Cited by 48 publications

(66 citation statements)

References 31 publications

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“…In contrast, maximal EROD activity of the CYP1A2 system increased to a maximum at 200 mM HEPES and declined with further increases in buffer concentration. Declining activities with increasing buffer concentrations were expected and have been attributed to the disruption of electrostatic interactions between reductase and P450, consistent with previous studies (6)(7)(8)(9)(10)(11)(12)(13)(14)16,17,24,39). When comparing EROD in the mixed reconstituted system to the sum of the rates of the binary systems (which can be seen by comparing the third and fourth bars from each group), a significant synergistic stimulation of EROD was observed at lower ionic strength; higher concentrations of HEPES relieved this synergism.…”

Section: Resultssupporting

confidence: 91%

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

2006

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Mixed reconstituted systems containing CYP2B4, CYP1A2 and NADPH-cytochrome P450 reductase were previously shown to exhibit a dramatic inhibition of 7-pentoxyresorufin-Odealkylation (PROD) when compared to simple reconstituted systems containing reductase and a single P450 enzyme, results consistent with the formation of CYP1A2-CYP2B4 complexes where the reductase binds with high affinity to the CYP1A2 moiety of the complex. In this report, we provide evidence for an interaction between CYP1A2 and CYP2E1. Synergism of 7-ethoxyresorufin-Odeethylation (EROD) and PROD was observed when these P450s were combined in mixed reconstituted systems at subsaturating reductase concentrations. Higher ionic strength attenuated the synergistic stimulation of both PROD and EROD in mixed reconstituted systems, consistent with disruption of heteromeric CYP2E1-CYP1A2 complexes. The effect of ionic strength was further examined as a function of reductase concentration. At lower ionic strength, there was a significant synergistic stimulation of EROD. This synergistic stimulation diminished with increasing reductase concentration, resulting in an additive response as reductase became saturating. Interestingly, at high ionic strength, the synergism of EROD in the mixed reconstituted system was not observed. In contrast, mixed reconstituted systems containing CYP2E1 and CYP2B4 did not provide evidence for the formation of these heteromeric P450-P450 complexes. The synergistic stimulation observed with the reductase-CYP1A2-CYP2E1 mixed reconstituted system is consistent with the formation of a CYP1A2-CYP2E1 complex. Taken together with the lack of a kinetically detectable interaction between CYP2B4 and CYP2E1, and the previously reported CYP1A2-CYP2B4 interaction, these results suggest that CYP1A2 may facilitate the formation of complexes with other P450 enzymes.The cytochrome P450 (P450) superfamily of proteins is functionally diverse, and well distributed in nature. In addition to their roles in the metabolism of numerous endogenous compounds, P450s are important contributors to drug metabolism, primarily by catalyzing the insertion of an oxygen atom into a substrate molecule. NADPH cytochrome P450 reductase (reductase) is the major electron transfer partner, and is required for the majority of NADPHdependent oxidation reactions. Multiple forms of cytochrome P450 exist in the endoplasmic reticulum in a large excess over reductase (1), with the ratio of P450 to reductase dependent † These studies were supported by a US Public Health Service Research Grant from the National Institute of Enviromental Health Sciences ES004344 (WLB), and a predoctoral fellowship from the Stanley Scott Cancer Center (RWK). * Correspondence should be addressed to: Wayne L. Backes, Ph.D., Department of Pharmacology and the Stanley S. Scott Cancer Center, LSU Health Sciences Center, 533 Bolivar Street, New Orleans, La 70112, Voice 504-568-6557, FAX 504-568-6888, emailwbacke@lsuhsc.edu SUPPORTING INFORMATION AVAILABLE The supporting information includes ...

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Section: Resultssupporting

confidence: 91%

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

2006

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“…Kelley et al (2005) used high concentrations of buffer salts to eliminate any ionic interactions between CYP1A2-CYP2B4 and were able to regain most of the activity that had been diminished by the purported interactions. However, circular dichroism studies have demonstrated conformational changes in proteins at high salt concentrations (Yun et al, 1996), rendering the aforementioned studies somewhat ambiguous. It is possible that ionic interactions play a role in P450-P450 interactions but additional work in this area is needed.…”

Section: Effect Of Cpr On 1:1 Cyp2c9/cyp2d6-mediated (S)-flurbiprofenmentioning

confidence: 99%

CYP2D6-CYP2C9 Protein-Protein Interactions and Isoform-Selective Effects on Substrate Binding and Catalysis

Subramanian

Löw²,

Locuson³

et al. 2009

Drug Metabolism and Disposition

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ABSTRACT:Cytochrome P450 (P450) protein-protein interactions have been observed with various in vitro systems. It is interesting to note that these interactions seem to be isoform-dependent, with some combinations producing no effect and others producing increased or decreased catalytic activity. With some exceptions, most of the work to date has involved P450s from rabbit, rat, and other animal species, with few studies including human P450s. In the studies presented herein, the interactions of two key drug-metabolizing enzymes, CYP2C9 and CYP2D6, were analyzed in a purified, reconstituted enzyme system for changes in both substrate-binding affinity and rates of catalysis. In addition, an extensive study was conducted as to the "order of mixing" for the reconstituted enzyme system and the impact on the observations. CYP2D6 coincubation inhibited CYP2C9-mediated (S)-flurbiprofen metabolism in a protein concentration-dependent manner. V max values were reduced by up to 50%, but no appreciable effect on K m was observed. Spectral binding studies revealed a 20-fold increase in the K S of CYP2C9 toward (S)-flurbiprofen in the presence of CYP2D6. CYP2C9 coincubation had no effect on CYP2D6-mediated dextromethorphan O-demethylation. The order of combination of the proteins (CYP2C9, CYP2D6, and cytochrome P450 reductase) influenced the magnitude of catalysis inhibition as well as the ability of increased cytochrome P450 reductase to attenuate the change in activity. A simple model, congruent with current results and those of others, is proposed to explain oligomer formation. In summary, CYP2C9-CYP2D6 interactions can alter catalytic activity and, thus, influence in vitro-in vivo correlation predictions.Cytochrome P450s (P450s) are responsible for the metabolism of more than 75% of the drugs on the market (Guengerich, 2006). Multiple subfamilies of cytochrome P450s can activate and metabolize a wide spectrum of substrates by a two-electron transfer catalytic cycle, in which electrons are provided by either NADPH-cytochrome P450 reductase (CPR) or cytochrome b 5 (b5), depending on the stage of the cycle (Guengerich, 2001). A number of studies have investigated the role of CPR and b5 and the nature of P450, CPR, and b5 interactions. Although CPR is indispensable for metabolism, cytochrome b 5 either has no effect or significantly enhances metabolism (Shimada et al., 1994;Locuson et al., 2006). Yamazaki et al. (1997) demonstrated that b5 stimulated the metabolism of tolbutamide and (S)-warfarin by CYP2C10 but had no effect on bufuralol hydroxylation by CYP1A1 and CYP2D6. In addition, both apo-and holo-b5 enhanced metabolism by CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5 in reconstituted systems (Shimada et al., 1994), suggesting that the effect of these protein-protein interactions of the redox partner with P450 may involve enzyme conformational changes or other mechanisms besides just provision of electrons.In addition to interactions with CPR and b5, P450s can also interact with each other, resulting in c...

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“…For the assay, two different approaches were taken to examine the effect of phospholipid and detergent on the P450 1A2 activity using the method described elsewhere with slight modification (14). For the NADPH-P450 reductase-supported reactions, 0.5 nmol of P450 1A2, 0.6 nmol of NADPH-P450 reductase, 15 g of phospholipid or detergent, and an NADPH-generating system were mixed together.…”

Section: Methodsmentioning

confidence: 99%

“…It was demonstrated that the stimulated activity of P450 1A2 by the high concentration of salt accompanies structural change of P450 1A2 in the presence of phospholipid (14). It seems worthwhile, therefore, to study the structural changes of P450 1A2 induced by phospholipid in detail to elucidate the relationship between the structure and the activity.…”

mentioning

confidence: 99%

Conformational Change of Cytochrome P450 1A2 Induced by Phospholipids and Detergents

Yun¹,

Song²,

Kim³

1997

Journal of Biological Chemistry

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Recently, it was reported that the activity of rabbit P450 1A2 is markedly increased at elevated salt concentration (Yun, C-H., Song, M., Ahn, T., and Kim, H. (1996) J. Biol. Chem. 271, 31312-31316). The activity increase of P450 1A2 coincides with the raised ␣-helix content and decreased ␤-sheet content. The presence of phospholipid magnified this effect. Here, possible structural change of rabbit P450 1A2 accompanying the phospholipid-induced increase in its enzyme activity was investigated by circular dichroism, fluorescence spectroscopy, and absorption spectroscopy. Studies with the reconstituted system supported by cumene hydroperoxide or NADPH showed that the P450 1A2 activities were found to be dependent on the head group and hydrocarbon chain length of phospholipid. Phosphatidylcholines having short hydrocarbon chains with a carbon number of 8 -12 were very efficient for reconstitution of the P450-catalyzed reactions supported by both cumene hydroperoxide and NADPH. It was found that the phospholipid increased the ␣-helix content and lowered the ␤-sheet content of P450. Intrinsic fluorescence intensity is also increased in the presence of phospholipid. The low spin iron configuration of P450 1A2 shifted toward the high spin configuration by most of the phospholipids in the endoplasmic reticulum. Some synthetic phospholipids having short hydrocarbon chains with a carbon number of 10 -12 caused a shift in the spin equilibrium of P450 1A2 toward low spin. The effect of detergents on the activity and conformation of P450 1A2 was also studied. It was found that the addition of detergents to P450 1A2 solution increased the enzyme activity of P450 1A2. Detergents also increased the ␣-helix content and lowered the ␤-sheet content of P450 1A2. Intrinsic fluorescence emissions also increased with the presence of detergents. Octyl glucoside and deoxycholate caused a shift toward high spin. On the other hand, cholate caused a shift toward low spin. It was found that the activity increase of rabbit P450 1A2 coincides with the conformational change including raised ␣-helix content. It is proposed that the interaction with the phospholipid molecules surrounding P450 1A2 in the endoplasmic reticulum is important for a functional conformation of P450 1A2 in a monooxygenase system including NADPH-P450 reductase.

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Conformational Change of Cytochrome P450 1A2 Induced by Sodium Chloride

Cited by 48 publications

References 31 publications

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

CYP2D6-CYP2C9 Protein-Protein Interactions and Isoform-Selective Effects on Substrate Binding and Catalysis

Conformational Change of Cytochrome P450 1A2 Induced by Phospholipids and Detergents

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