TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROME<i>B</i><sub>5</sub>AND OF SITE-DIRECTED MUTAGENESIS

Yamaguchi, Yoshitaka; Khan, Kishore K.; He, You-Ai; He, Yeping; Halpert, James R.

doi:10.1124/dmd.32.1.155

Cited by 21 publications

(31 citation statements)

References 28 publications

(31 reference statements)

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“…These results suggested that the inactivation of P450 2B6 was not due to heme destruction but to covalent modification of the apoprotein. In addition, incubation of the trifluoroaryldiaziridines with recombinant P450 2B6 did not result in the appearance of a peak at 478 nm or a decrease in the native heme peak at 418 nm as would be expected for a phenyl-iron complex (data not shown) (Raag et al, 1990;Yamaguchi et al, 2004) Extensive dialysis of P450 2B6 after inactivation by the five trifluoroaryldiaziridines did not result in recovery of any of the enzyme activity, suggesting that the reactive species formed during the inactivation covalently modified the enzyme. To verify that the loss in enzymatic activity resulted from covalent modification of P450 2B6 and not modification of reductase, fresh reductase was added to the dialyzed samples and the samples were checked for P450 catalytic activity.…”

Section: Discussionmentioning

confidence: 89%

Synthesis of Substituted Phenyl Diaziridines and Characterization as Mechanism-Based Inactivators of Human Cytochrome P450 2B6

et al. 2006

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ABSTRACT:The metabolism of arylhydrazines by cytochromes P450 (P450s) has previously been shown to yield aryl-iron complexes that inhibit P450 enzymes as a result of heme modification. These modifications of the heme have been used to probe the topology of the active site of several P450s. Therefore, diaziridines containing one or more substitutions on the phenyl ring were synthesized and evaluated as potential mechanism-based inactivators of P450 2B enzymes that could be used to elucidate the active site topology.Five of the six trifluoroaryldiaziridines tested selectively inactivated P450 2B6 in the reconstituted system in a time-, concentration-, and NADPH-dependent manner as measured using the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation assay. The kinetic parameters for P450 2B6 inactivation by the five compounds were calculated. Analysis of the P450 heme from P450s inactivated by the five substituted diaziridines suggested that the activity loss was not due to heme destruction as measured by the reduced-CO spectrum or high-performance liquid chromatography of the P450 heme. Dialysis experiments indicated the irreversible nature of the inactivation and the reaction between the diaziridine compounds and the P450 enzyme. Interestingly, a thiomethyl-substituted phenyl diaziridine had no effect on the activity of P450 2B6 in the reconstituted system, but competitively inhibited the O-debenzylation activity of P450 3A4 with 7-benzyloxy-4-(trifluoromethyl)coumarin as substrate. Binding spectra suggest that this compound bound reversibly to P450 2B6, and preliminary results indicate that 3-(4-methylthiophenyl)-3-(trifluoromethyl)diaziridine is metabolized by P450 2B6.

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Section: Discussionmentioning

confidence: 89%

Synthesis of Substituted Phenyl Diaziridines and Characterization as Mechanism-Based Inactivators of Human Cytochrome P450 2B6

et al. 2006

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“…Indeed, the redox partners such as cytochrome b 5 and CPR can also affect allostery by binding at the redox partner binding site of the CYP molecule and inducing changes in the CYP conformation. Clearly, the subtle structural alterations of the active site of CYP3A4 isoform during attack of phenyldiazene, through the availability of nitrogen atoms of the haem cofactor, was evident with the modulation of the activity in the presence of both cytochrome b 5 and CPR (Yamaguchi et al 2004).…”

Section: Mechanism Of Stimulation Of Cyp17 Cleavage Activitymentioning

confidence: 99%

Cytochrome b5 modulation of 17α hydroxylase and 17–20 lyase (CYP17) activities in steroidogenesis

Akhtar¹,

Kelly²,

Kaderbhai³

2005

Journal of Endocrinology

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CYP17 is a steroidogenic enzyme located in the zona fasciculata and zona reticularis of the adrenal cortex and gonad tissues and which has dual functions -hydroxylation and as a lyase. The first activity gives hydroxylation of pregnenolone and progesterone at the C 17 position to generate 17 -hydroxypregnenolone and 17 -hydroxyprogesterone, while the second enzymic activity cleaves the C 17 -C 20 bond of 17 -hydroxypregnenolone and 17 -hydroxyprogesterone to form dehydroepiandrosterone and androstenedione respectively. The modulation of these two activities occurs through cytochrome b 5 . Association of cytochrome b 5 and CYP17 is thought to be based primarily on electrostatic interactions in which the negatively charged residues pair up with positively charged residues on the proximal surface of the CYP17 molecule. Non-specific interactions of the hydrophobic membrane regions of cytochrome b 5 and CYP17 are also thought to play a crucial role in the association of these two haemoproteins. Although cytochrome b 5 is known to stimulate CYP activity by contributing the second electron in the catalytic cycle, in the case of CYP17, the mechanism of cleavage stimulation proceeds via an allosteric mode. It is hypothesised that cytochrome b 5 promotes the cleavage by aligning the iron-oxygen complex attack onto the C 20 rather than the C 17 atom of the steroid substrate molecule. Thus, further understanding of the mechanism of modulation by cytochrome b 5 of the hydroxylase and lyase activities should shed new insights on developing therapeutic targets in CYP17-linked biochemical processes such as adrenarche, polycystic ovary syndrome and prostate cancer.

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“…42 a domain which flanks the heme group and contains a substrate recognition site (SRS4). 58 As shown in the Supplementary Table S2, Tyr319 is a highly conserved residue in the cytochrome P450 family throughout eukaryote evolution from nematode to human. In order to predict the functional impact of the amino acid mutation, we used two in silico prediction algorithms, PolyPhen and PANTHER, which combine protein structure and sequence evolution analysis to achieve a more comprehensive result.…”

Section: Resultsmentioning

confidence: 99%

Analysis of CYP3A4 genetic polymorphisms in Han Chinese

Zhou

Shu

et al. 2011

J Hum Genet

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Our study aimed to comprehensively investigate the genetic polymorphisms of CYP3A4 in Han Chinese. We sequenced the gene regions of CYP3A4, including its promoter, exons, surrounding introns and 3¢ untranslated region (3¢UTR), from 100 unrelatedhealthy Han Chinese individuals. We detected 11 SNPs, three of which are novel. According to in silico functional prediction of novel variants, 20148 A4G in exon 10, resulting in substitution of Tyr319 with Cys (CYP3A4*21), may induce dramatic alteration of protein conformation, and 26908 G4A in 3¢UTR may disrupt post-transcriptional regulation. We identified five alleles in Han Chinese, the allele frequencies of CYP3A4*1, *5, *6, *18 and *21 are 97, 0.5, 1, 1 and 0.5%, respectively. Haplotype inference revealed 14 haplotypes, of which the major haplotype CYP3A4*1A constitutes 59% of the total chromosomes. We also examined the possible role of natural selection in shaping the variation of CYP3A4 and confirmed a trend, consistent with the action of positive selection. We systematically screened the genetic polymorphisms of CYP3A4 in Han Chinese, highlighted possible functional impairment of the novel allele and summarized the distinct allele and haplotype frequency distribution, with an emphasis on detecting the footprint of recent positive selection on the CYP3A4 gene in Han Chinese.

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TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROMEB₅AND OF SITE-DIRECTED MUTAGENESIS

Abstract: This article is available online at http://dmd.aspetjournals.org ABSTRACT:The active site topology of heterologously expressed CYP3A4 purified from an Escherichia coli expression system was examined using phenyldiazene.

Cited by 21 publications

References 28 publications

Synthesis of Substituted Phenyl Diaziridines and Characterization as Mechanism-Based Inactivators of Human Cytochrome P450 2B6

Synthesis of Substituted Phenyl Diaziridines and Characterization as Mechanism-Based Inactivators of Human Cytochrome P450 2B6

Cytochrome b5 modulation of 17α hydroxylase and 17–20 lyase (CYP17) activities in steroidogenesis

Analysis of CYP3A4 genetic polymorphisms in Han Chinese

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TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROMEB5AND OF SITE-DIRECTED MUTAGENESIS

Abstract: This article is available online at http://dmd.aspetjournals.org ABSTRACT:The active site topology of heterologously expressed CYP3A4 purified from an Escherichia coli expression system was examined using phenyldiazene.

Cited by 21 publications

References 28 publications

Synthesis of Substituted Phenyl Diaziridines and Characterization as Mechanism-Based Inactivators of Human Cytochrome P450 2B6

Synthesis of Substituted Phenyl Diaziridines and Characterization as Mechanism-Based Inactivators of Human Cytochrome P450 2B6

Cytochrome b5 modulation of 17α hydroxylase and 17–20 lyase (CYP17) activities in steroidogenesis

Analysis of CYP3A4 genetic polymorphisms in Han Chinese

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TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROMEB₅AND OF SITE-DIRECTED MUTAGENESIS