Substrate binding to cytochromes P450

Isin, Emre M.; Guengerich, F. Peter

doi:10.1007/s00216-008-2244-0

Cited by 111 publications

(93 citation statements)

References 188 publications

(222 reference statements)

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“…From these results, it would be reasonable to consider that UGT2B7 suppresses CYP3A4 activity by inhibiting the insertion of substrates into the catalytic pocket of CYP3A4. The binding of substrate to P450 causes a change in its electrical potential, and this change drives the catalytic cycle of P450 (Isin and Guengerich, 2008). If UGT2B7 inhibits this first step, CYP3A4 function would be suppressed in all the later catalytic steps as we observed.…”

Section: Ugt2b7 Suppresses Cyp3a4 Functionsupporting

confidence: 53%

Suppression of Cytochrome P450 3A4 Function by UDP-Glucuronosyltransferase 2B7 through a Protein-Protein Interaction: Cooperative Roles of the Cytosolic Carboxyl-Terminal Domain and the Luminal Anchoring Region

Miyauchi

Nagata

Yamazoe³

et al. 2015

Mol Pharmacol

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There is a large discrepancy between the interindividual difference in the hepatic expression level of cytochrome P450 3A4 (CYP3A4) and that of drug clearance mediated by this enzyme. However, the reason for this discrepancy remains largely unknown. Because CYP3A4 interacts with UDP-glucuronosyltransferase 2B7 (UGT2B7) to alter its function, the reverse regulation is expected to modulate CYP3A4-catalyzed activity. To address this issue, we investigated whether protein-protein interaction between CYP3A4 and UGT2B7 modulates CYP3A4 function. For this purpose, we coexpressed CYP3A4, NADPH-cytochrome P450 reductase, and UGT2B7 using a baculovirus-insect cell system. The activity of CYP3A4 was significantly suppressed by coexpressing UGT2B7, and this suppressive effect was lost when UGT2B7 was replaced with calnexin (CNX). These results strongly suggest that UGT2B7 negatively regulates CYP3A4 activity through a protein-protein interaction. To identify the UGT2B7 domain associated with CYP3A4 suppression we generated 12 mutants including chimeras with CNX. Mutations introduced into the UGT2B7 carboxylterminal transmembrane helix caused a loss of the suppressive effect on CYP3A4. Thus, this hydrophobic region is necessary for the suppression of CYP3A4 activity. Replacement of the hydrophilic end of UGT2B7 with that of CNX produced a similar suppressive effect as the native enzyme. The data using chimeric protein demonstrated that the internal membrane-anchoring region of UGT2B7 is also needed for the association with CYP3A4. These data suggest that 1) UGT2B7 suppresses CYP3A4 function, and 2) both hydrophobic domains located near the C terminus and within UGT2B7 are needed for interaction with CYP3A4.

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Section: Ugt2b7 Suppresses Cyp3a4 Functionsupporting

confidence: 53%

Suppression of Cytochrome P450 3A4 Function by UDP-Glucuronosyltransferase 2B7 through a Protein-Protein Interaction: Cooperative Roles of the Cytosolic Carboxyl-Terminal Domain and the Luminal Anchoring Region

Miyauchi

Nagata

Yamazoe³

et al. 2015

Mol Pharmacol

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“…Co-crystallization of progesterone with 3A4 identified a peripheral binding site for the steroid near the outer surface of helices FЈ and GЈ and the phenylalanine cluster (68). 3A4 often exhibits homo-and heterotropic activation kinetics with progesterone and other substrates of similar size that are consistent with the binding of two or more molecules to the enzyme (73,74). Biophysical studies have suggested that two molecules can stack in the active site (75), as seen for the 3A4 structure with two molecules of ketoconazole (69), and have provided evidence for a peripheral binding site such as that observed for progesterone (76).…”

Section: Drug-metabolizing Enzymesmentioning

confidence: 82%

Structural Diversity of Eukaryotic Membrane Cytochrome P450s

Johnson¹,

Stout

2013

Journal of Biological Chemistry

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X-ray crystal structures are available for 29 eukaryotic microsomal, chloroplast, or mitochondrial cytochrome P450s, including two non-monooxygenase P450s. These structures provide a basis for understanding structure-function relations that underlie their distinct catalytic activities. Moreover, structural plasticity has been characterized for individual P450s that aids in understanding substrate binding in P450s that mediate drug clearance.

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“…Binding of substrates and xenobiotics to P450s can result in two types of characteristic spectral changes in the UV/VIS heme Soret spectrum, referred to as Type I and Type II (Schenkman et al, 1981;Isin and Guengerich, 2008). A classical Type I spectrum results from the spin-state shift of the Soret band at about 418 nm that represents the ferric (Fe III ) low-spin substrate free form tõ 390 nm for the ferric (Fe III ) high-spin form originating from the displacement of H 2 O as the sixth ligand when a substrate is bound in close proximity to the heme.…”

Section: Determination Of Spectral Dissociation Constantsmentioning

confidence: 99%

“…A classical Type I spectrum results from the spin-state shift of the Soret band at about 418 nm that represents the ferric (Fe III ) low-spin substrate free form tõ 390 nm for the ferric (Fe III ) high-spin form originating from the displacement of H 2 O as the sixth ligand when a substrate is bound in close proximity to the heme. Direct coordination of a ligand to heme iron results in a Type II spectrum characterized by a shift to 430-455 nm, but these complexes are inhibitory and generally not considered relevant to productive substrate binding leading to catalysis (Isin and Guengerich, 2008).…”

Section: Determination Of Spectral Dissociation Constantsmentioning

confidence: 99%

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

Roellecke

Jäger

Gyurov

et al. 2017

Protein Engineering, Design and Selection

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Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wildtype rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l −1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.

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Substrate binding to cytochromes P450

Cited by 111 publications

References 188 publications

Suppression of Cytochrome P450 3A4 Function by UDP-Glucuronosyltransferase 2B7 through a Protein-Protein Interaction: Cooperative Roles of the Cytosolic Carboxyl-Terminal Domain and the Luminal Anchoring Region

Suppression of Cytochrome P450 3A4 Function by UDP-Glucuronosyltransferase 2B7 through a Protein-Protein Interaction: Cooperative Roles of the Cytosolic Carboxyl-Terminal Domain and the Luminal Anchoring Region

Structural Diversity of Eukaryotic Membrane Cytochrome P450s

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

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