Catalytic turnover of pyrene by CYP3A4: Evidence that cytochrome b5 directly induces positive cooperativity

Jushchyshyn, Monica I.; Hutzler, J. Matthew; Schrag, Michael; Wienkers, Larry C.

doi:10.1016/j.abb.2005.02.027

Cited by 42 publications

(23 citation statements)

References 33 publications

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“…4B), i.e. described with a low K m /high K m fit (not sigmoidal) (28), and the patterns were altered in the presence of cytochrome b 5 . Benzo[a]pyrene, which contains one more aromatic ring than pyrene, has been studied extensively in the context of chemical carcinogenesis (86).…”

Section: Discussionmentioning

confidence: 99%

“…However, in the 1-hydroxylation of pyrene by P450 3A4 only weak homotropic cooperativity was observed, with an apparent Hill coefficient (n) of ϳ1.7 in some cases, i.e. with cytochrome b 5 (28,87). One possibility is that some cases of positive and negative cooperativity in the P450s can be explained by models such as that described in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…A full discussion of the field is beyond the scope of this introduction and the reader is referred to some of the more recent reviews (4,17,18). Some of the major features of the more common proposals include the following: (i) a classic allosteric reaction with a second ligand-binding site controlling binding to the catalytic substrate-binding site (22,23); (ii) cooccupancy of the cognate substrate-binding site with multiple ligands, leading to a more productive rearrangement of the substrate for catalysis because of either steric packing or more specific protein-ligand interactions (11,24,25); (iii) selective populations of P450 (in slow equilibrium) that interact in different ways (26); (iv) multimeric states of P450 (27) and possibly interactions with accessory proteins (28) that are involved; and (v) elements of an induced fit process, in which the protein adapts a conformation as a result of an initial interaction with a ligand (29). Some of these possibilities are not mutually exclusive.…”

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confidence: 99%

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Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2

Sohl

Isin

Eoff

et al. 2008

Journal of Biological Chemistry

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Rabbit liver cytochrome P450 (P450) 1A2 was found to catalyze the 5,6-epoxidation of ␣-naphthoflavone (␣NF), 1-hydroxylation of pyrene, and the subsequent 6-, 8-, and other hydroxylations of 1-hydroxy (OH) pyrene. Plots of steady-state rates of product formation versus substrate concentration were hyperbolic for ␣NF epoxidation but highly cooperative (Hill n coefficients of 2-4) for pyrene and 1-OH pyrene hydroxylation. When any of the three substrates (␣NF, pyrene, 1-OH pyrene) were mixed with ferric P450 1A2 using stopped-flow methods, the changes in the heme Soret spectra were relatively slow and multiphasic. Changes in the fluorescence of all of the substrates were much faster, consistent with rapid initial binding to P450 1A2 in a manner that does not change the heme spectrum. For binding of pyrene to ferrous P450 1A2, the course of the spectra revealed sequential changes in opposite directions, consistent with P450 1A2 being involved in a series of transitions to explain the kinetic multiphasicity as opposed to multiple, slowly interconverting populations of enzyme undergoing the same event at different rates. Models of rabbit P450 1A2 based on a published crystal structure of a human P450 1A2-␣NF complex show active site space for only one ␣NF or for two pyrenes. The spectral changes observed for binding and hydroxylation of pyrene and 1-OH pyrene could be fit to a kinetic model in which hydroxylation occurs only when two substrates are bound. Elements of this mechanism may be relevant to other cases of P450 cooperativity.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2

Sohl

Isin

Eoff

et al. 2008

Journal of Biological Chemistry

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“…As has been shown for other cytochromes P450 (P450s) such as CYP2C8 (Kaspera et al, 2011) and CYP2C9 (Kumar et al, 2006), several factors inherent to specific enzyme sources that include differences in cytochrome b5 (Cyt b5) contents may influence in vitro kinetic parameters and inhibition constants in a substrate-dependent manner. Cyt b5 has been reported to activate several P450s including CYP2B6 (Reed and Hollenberg, 2003;Jushchyshyn et al, 2005), but its influence on the catalytic properties of CYP2B6.6 protein has not been studied. Therefore, the second purpose was to test the influence of Cyt b5 on metabolic activities of expressed CYP2B6.1 and CYP2B6.6 proteins.…”

Section: Introductionmentioning

confidence: 99%

Effects of the CYP2B6*6 Allele on Catalytic Properties and Inhibition of CYP2B6 In Vitro: Implication for the Mechanism of Reduced Efavirenz Metabolism and Other CYP2B6 Substrates In Vivo

Xu¹,

Ogburn²,

Guo³

et al. 2012

Drug Metabolism and Disposition

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ABSTRACT:The mechanism by which CYP2B6*6 allele alters drug metabolism in vitro and in vivo is not fully understood. To test the hypothesis that altered substrate binding and/or catalytic properties contribute to its functional consequences, efavirenz 8-hydroxylation and bupropion 4-hydroxylation were determined in CYP2B6.1 and CYP2B6.6 proteins expressed without and with cytochrome b5 (Cyt b5) and in human liver microsomes (HLMs) obtained from liver tissues genotyped for the CYP2B6*6 allele. The susceptibility of the variant protein to inhibition was also tested in HLMs. Significantly higher V max and K m values for 8-hydroxyefavirenz formation and ϳ2-fold lower intrinsic clearance (Cl int ) were noted in expressed CYP2B6.6 protein (؊b5) compared with that of CYP2B6.1 protein (؊b5); this effect was abolished by Cyt b5. The V max and Cl int values for 4-hydroxybupropion formation were significantly higher in CYP2B6.6 than in CYP2B6.1 protein, with no difference in K m , whereas coexpression with Cyt b5 reversed the genetic effect on these kinetic parameters. In HLMs, CYP2B6*6/*6 genotype was associated with markedly lower V max (and moderate increase in K m ) and thus lower Cl int values for efavirenz and bupropion metabolism, but no difference in catalytic properties was noted between CYP2B6*1/*1 and CYP2B6*1/*6 genotypes. Inhibition of efavirenz 8-hydroxylation by voriconazole was significantly greater in HLMs with the CYP2B6*6 allele (K i ‫؍‬ 1.6 ؎ 0.8 M) than HLMs with CYP2B6*1/*1 genotype (K i ‫؍‬ 3.0 ؎ 1.1 M). In conclusion, our data suggest the CYP2B6*6 allele influences metabolic activity by altering substrate binding and catalytic activity in a substrate-and Cyt b5-dependent manner. It may also confer susceptibility to inhibition.

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“…The addition of cyt b5 to mixtures of P450s and CPR results in varied effects on the activity of P450-mediated drug oxidation. There are reports of increases , decreases (Morgan and Coon, 1984;Gruenke et al, 1995), or no effect on the rate of drug metabolism, and changes in kinetic profiles (Jushchyshyn et al, 2005) induced by cyt b5, all of which appear to be P450 isoform-and substratedependent. Any correlation between reconstituted systems and more complex systems, such as microsomes, requires an understanding of how the different constituents in the system interact.…”

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confidence: 99%

CYP2C9 Protein Interactions with Cytochrome b5: Effects on the Coupling of Catalysis

Locuson¹,

Wienkers²,

Jones³

et al. 2007

Drug Metabolism and Disposition

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ABSTRACT:The hemoprotein cytochrome b 5 (cyt b5) has been demonstrated to affect the kinetics of drug oxidation by the microsomal cytochromes P450 (P450s). However, the mechanisms through which cyt b5 exerts these effects are variable and P450 isoform-dependent. Whereas the effects of cyt b5 on the major drug-metabolizing enzymes CYP2D6, CYP2E1, and CYP3A4 are well studied, fewer studies conducted over limited ranges of cyt b5 concentrations have been performed on CYP2C9. In the present study with CYP2C9, cyt b5 exerted complex actions upon P450 oxidative reactions by affecting the rate of metabolite formation, the consumption of NADPH by cytochrome P450 reductase, and uncoupling of the reaction cycle to hydrogen peroxide and water. Cytochrome b 5 devoid of the heme moiety (apo-b5) exhibited effects similar to those of native cyt b5. All rates were highly dependent on the cyt b5 to CYP2C9 enzyme ratio, suggesting that the amount of cyt b5 present in an in vitro incubation is an important factor that can have an impact on the reliability of extrapolating in vitro generated data to predict the in vivo condition. The major effects of cyt b5 are hypothesized to result from a cyt b5-induced conformational change in CYP2C9 that results in an increased collision frequency between the iron-oxygen species (Cpd I) and the substrate, and a decrease in the oxidase activity. Together, these findings suggest that cyt b5 can alter multiple steps in the P450 catalytic cycle via complex interactions with P450 and P450 reductase.

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Catalytic turnover of pyrene by CYP3A4: Evidence that cytochrome b5 directly induces positive cooperativity

Cited by 42 publications

References 33 publications

Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2

Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2

Effects of the CYP2B6*6 Allele on Catalytic Properties and Inhibition of CYP2B6 In Vitro: Implication for the Mechanism of Reduced Efavirenz Metabolism and Other CYP2B6 Substrates In Vivo

CYP2C9 Protein Interactions with Cytochrome b5: Effects on the Coupling of Catalysis

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