Allosteric phenomena in cytochrome P450‐catalyzed monooxygenations

Hlavica, Peter; Lewis, David F.

doi:10.1046/j.1432-1327.2001.02412.x

Cited by 70 publications

(47 citation statements)

References 167 publications

(246 reference statements)

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“…The K m and V max values for MR were not markedly different between BCN and wild type, whereas for BFC there was a 5-fold increase in K m and a 4.5-fold decrease in V max in the BCN samples ( Table 2). The alteration in K m for BFC provides evidence to support theory that cytochrome b 5 effects P450 activity through allosteric interactions with the metabolizing cytochrome P450s, causing a conformational shift in the active site, which affects substrate binding (Bridges et al, 1998;Hlavica and Lewis, 2001). Alternatively, the change in K m observed for BFC could be attributable to multiple P450s, differentially up-regulated by the deletion of cytochrome b 5 contributing to the metabolism of this substrate.…”

Section: Discussionmentioning

confidence: 54%

Deletion of Microsomal Cytochrome b5 Profoundly Affects Hepatic and Extrahepatic Drug Metabolism

McLaughlin

Ronseaux²,

Finn

et al. 2010

Molecular Pharmacology

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We demonstrated recently that cytochrome b 5 plays an important in vivo role in hepatic cytochrome P450 (P450) function [J Biol Chem 283:31385-31393, 2008]. We have now generated a model in which cytochrome b 5 has been deleted in all tissues [cytochrome b 5 complete null (BCN)], which surprisingly results in a viable mouse despite the putative in vivo roles of this protein in lipid and steroid hormone metabolism and the reduction of methemoglobin. In contrast to the liver-specific deletion, complete deletion of cytochrome b 5 leads to a neonatal increase in the expression of many hepatic P450s at both the protein and mRNA level. In extrahepatic tissues, some changes in P450 expression were also observed that were isoformdependent. In vitro cytochrome P450 activities in liver, kidney, lung, and small intestine of BCN mice were determined for a range of model substrates and probe drugs; a profound reduction in the metabolism of some substrates, particularly in lung, kidney, and small intestine, was observed. In vivo, the metabolism of metoprolol was significantly altered in BCN mice, in contrast to the previous finding in the liver-specific cytochrome b 5 deletion, suggesting that extrahepatic cytochrome b 5 plays a significant role in its disposition. Testicular Cyp17 hydroxylase and lyase activities were also significantly reduced by cytochrome b 5 deletion, leading to significantly lower levels of testicular testosterone. The BCN mouse provides an additional model system with which to further investigate the functions of cytochrome b 5 , particularly in extrahepatic tissues.

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Section: Discussionmentioning

confidence: 54%

Deletion of Microsomal Cytochrome b5 Profoundly Affects Hepatic and Extrahepatic Drug Metabolism

McLaughlin

Ronseaux²,

Finn

et al. 2010

Molecular Pharmacology

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“…CYP3A4 displays both homotropic and heterotropic cooperativity, attributed variously to the binding of multiple substrate molecules within a single binding site or at distinct sites (43)(44)(45)(46). Although in this case direct interaction seems supported by the specificity of the effect (no cooperativity with a-or b-TOHs), a membrane-mediated effector mechanism of substrate or products (the latter of which likely remain in the membrane) cannot be ruled out (47). The positive effector feature of a-TOH, however, is probably not attributable to a general effect on membrane fluidity.…”

Section: Discussionmentioning

confidence: 98%

Influence of major structural features of tocopherols and tocotrienols on their ω-oxidation by tocopherol-ω-hydroxylase

Sontag

Parker

2007

Journal of Lipid Research

174

157

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Human cytochrome P450 4F2 (CYP4F2) catalyzes the initial v-hydroxylation reaction in the metabolism of tocopherols and tocotrienols to carboxychromanols and is, to date, the only enzyme shown to metabolize vitamin E. The objective of this study was to characterize this activity, particularly the influence of key features of tocochromanol substrate structure. The influence of the number and positions of methyl groups on the chromanol ring, and of stereochemistry and saturation of the side chain, were explored using HepG2 cultures and microsomal reaction systems. Human liver microsomes and microsomes selectively expressing recombinant human CYP4F2 exhibited substrate activity patterns similar to those of HepG2 cells. Although activity was strongly associated with substrate accumulation by cells or microsomes, substantial differences in specific activities between substrates remained under conditions of similar microsomal membrane substrate concentration. Methylation at C5 of the chromanol ring was associated with markedly low activity. Tocotrienols exhibited much higher V max values than their tocopherol counterparts. Side chain stereochemistry had no effect on v-hydroxylation of a-tocopherol (a-TOH) by any system. Kinetic analysis of microsomal CYP4F2 activity revealed Michaelis-Menten kinetics for a-TOH but allosteric cooperativity for other vitamers, especially tocotrienols. Additionally, a-TOH was a positive effector of v-hydroxylation of other vitamers. These results indicate that CYP4F2-mediated tocopherol-vhydroxylation is a central feature underlying the different biological half-lives, and therefore biopotencies, of the tocopherols and tocotrienols.-Sontag, T. J., and R. S. Parker. Influence of major structural features of tocopherols and tocotrienols on their v-oxidation by tocopherol-v-hydroxylase. J. Lipid Res. 2007. 48: 1090-1098. Supplementary key words vitamin EVitamin E is the generic term for the tocopherols and tocotrienols, which differ in the number and positions of methyl groups around the chromanol ring and in the saturation and stereochemistry of the phytyl tail (Fig. 1).These subtle structural variations are associated with substantial differences in their biopotency in vivo. Although a-tocopherol (a-TOH) has been studied most intensively, recent research has also suggested important roles for the non-a vitamers of vitamin E. Bioactivities of tocopherols and tocotrienols include the more familiar trapping of radical species, including nitric oxide (1-9), as well as more provocative activities, such as the regulation of gene transcription (10-13), anti-inflammation (14), inhibition of cholesterol synthesis (15), and apoptosis (16-18).The superior activity of RRR-a-TOH in vivo appears to result from its superior retention in the body relative to other forms of vitamin E (19, 20). Although an important factor involved in this selectivity is the hepatic atocopherol transfer protein (a-TTP) (21-24), differential rates of postabsorptive metabolism of the tocopherols and tocotrienols to w...

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“…Therefore, many recent efforts in the studies of P450 cooperativity are increasingly focused on the substrate binding stage of the catalytic cycle (16,17,(19)(20)(21)(22). In the case of Type-I substrates these interactions are known to modulate the spin equilibrium of the enzyme, which is thought to be an important determinant of its catalytic efficiency and coupling (23)(24)(25)(26)(27)(28). Homotropic cooperativity in substrate binding is revealed in sigmoidal dependencies of the substrate-induced spin transitions on the substrate concentration.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Resolution of Multiple Substrate Binding Sites in Cytochrome P450 3A4: The Stoichiometry of the Enzyme−Substrate Complexes Probed by FRET and Job's Titration

2006

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To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the Type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 ± 0.3 (S 50 = 8.0 ± 1.1 µM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting K D values of 0.31 ± 0.08 µM and 6.7 ± 1.9 µM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with K D values of 0.16 ± 0.09, 4.8 ± 1.4, and 0.18 ± 0.09 µM for 1-PB, PMA, and bromocriptine respectively. Correspondingly, the Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of K D obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.Keywords substrate binding; cooperativity; conformers; spin equilibrium Recent studies of function and regulation of cytochromes P450 reveal increasing attention to the mechanisms and pharmacological significance of homo-and heterotropic cooperativity observed in various mammalian P450 species (1-8). The most prominent examples of these phenomena are observed with cytochrome P450 3A4 (CYP3A4), the most abundant P450 in human liver. CYP3A4 is responsible for the metabolism of a broad range of drug substrates, and studies of cooperativity of this enzyme are of vital importance to our efforts to elucidate the mechanisms of drug metabolism in humans (9). However, despite extensive studies, a general mechanism of cooperativity remains obscure. † This research was supported by NIH grants GM54995 (JRH), and Center grant ES06676 (JRH). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe prevailing hypothesis is that cytochromes P450 exhibiting cooperativity accommodate multiple substrate molecules in one large binding pocket (10)(11)(12). A loose fit of a single substrate molecule requires the binding of a second ligand for efficient binding and/or catalysis (11-13). However, the possibility that P450 cooperativity reflects a true case of a...

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Allosteric phenomena in cytochrome P450‐catalyzed monooxygenations

Cited by 70 publications

References 167 publications

Deletion of Microsomal Cytochrome b5 Profoundly Affects Hepatic and Extrahepatic Drug Metabolism

Deletion of Microsomal Cytochrome b5 Profoundly Affects Hepatic and Extrahepatic Drug Metabolism

Influence of major structural features of tocopherols and tocotrienols on their ω-oxidation by tocopherol-ω-hydroxylase

Resolution of Multiple Substrate Binding Sites in Cytochrome P450 3A4: The Stoichiometry of the Enzyme−Substrate Complexes Probed by FRET and Job's Titration

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