Pyrene·Pyrene Complexes at the Active Site of Cytochrome P450 3A4:  Evidence for a Multiple Substrate Binding Site

Dabrowski, Michael J.; Schrag, Michael; Wienkers, Larry C.; Atkins, William M.

doi:10.1021/ja027552x

Cited by 91 publications

(65 citation statements)

References 19 publications

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“…Interestingly, the active site of P450 3A4 is known to be very large (20,21,29). Interactions among multiple ligands have been invoked as a mechanism for cooperativity with P450 3A4 (11), and stronger fluorescence evidence for the presence of pyrene excimers in the protein has been presented (73). 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.…”

Section: Discussionmentioning

confidence: 99%

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%

Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2

Sohl

Isin

Eoff

et al. 2008

Journal of Biological Chemistry

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“…Fluorescence spectroscopy has been successfully exploited by several labs to probe the various putative binding sites of CYP3A4 [10,12,16,17,22,40]. Some studies have utilized hemedependent quenching of fluorophores to measure ligand binding and to monitor putative conformational changes [22,23].…”

Section: Discussionmentioning

confidence: 99%

“…Most models used to explain the cooperativity of this enzyme have relied on the postulate that multiple ligands bind simultaneously to the enzyme [8,14,15], although direct confirmation of the specific location of binding sites has been challenging. Spectroscopic studies [16,17] and recently published X-ray crystal structures [18] have suggested that multiple ligand binding within the CYP3A4 active site is possible, as is binding at a remote site on the protein surface. However, allosteric kinetics could be observed, even in the absence of multiple ligand binding, if kinetically distinct conformations are not in rapid equilibrium [19,20].…”

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confidence: 99%

Spectral resolution of a second binding site for Nile Red on cytochrome P4503A4

Nath

Fernández

Lampe

et al. 2008

Archives of Biochemistry and Biophysics

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Nile Red is sequentially metabolized by Cytochrome P4503A4 to the N-monoethyl and N-desethyl products, which typifies the metabolism of many amine-containing drugs. Sequential metabolism of a single substrate results in complex kinetics that confound predictive models of drug clearance. As a fluorescent model for drugs which undergo sequential metabolism, Nile Red provides the opportunity to monitor drug-CYP interactions wherein the fluorescent properties of Nile Red could, in principle, be exploited to determine individual rate and equilibrium constants for the individual reactions. Previously, it was shown that Nile Red binds at the active site and fluoresces (K D = 50 nM) with maximum emission at ~620 nm, but it was unclear whether a red-shifted emission, at ~660 nm, consisted of only free Nile Red or Nile Red bound at a second site on the protein. Here, equilibrium binding studies, including 'reverse titrations' spanning low ratios of CYP3A4/Nile Red, indicate two binding sites for Nile Red with a contribution to the 'red emission' greater than can be accounted for by free Nile Red. Singular value decomposition affords basis spectra for both spectral components and fits well to the experimentally determined concentration dependence of Nile Red emission. In addition, the red spectral component, with an apparent K D = 2.2 µM, is selectively eliminated by titration with the known allosteric effectors of CYP3A4, α-napthoflavone and testosterone. Furthermore, the double mutant L2311F/D214E, previously demonstrated to perturb a peripheral allosteric site, lacks the red-emitting Nile Red binding site, but retains the blue-emitting site. Together these data indicate that a second Nile Red site competes with other effectors of CYP3A4 at a site that results in Nile Red emission at 660 nm. KeywordsCytochrome P450 3A4; drug metabolism; allosterism; homotropic effectors; Nile Red; solvatochromism As detoxification enzymes, Cytochrome P450's (CYPs) 1 catalyze a wide range of oxidative and reductive reactions with an enormous range of structurally distinct substrates [1][2][3][4]. CYPs dominate the metabolism of nearly all prescribed therapeutic drugs, as well as a number of endogenous hormones and xenobiotics, and thus control their steady state plasma and tissue levels [1,5,6]. Their ability to perform complex oxidative metabolism on a structurally diverse set of compounds is often accompanied by complex non-Michaelis-Menten, or "allosteric", kinetics [7][8][9]. The predominant human hepatic drug metabolizing CYP isoform, CYP 3A4, *e-mail: winky@u.washington.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaim...

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“…Although most analysis of P450 cooperativity has been based on steady-state kinetics of substrate oxidation, more recent attention has focused on investigating the substrate binding step in the CYP3A4 catalytic cycle [20,[40][41][42][43][44][45][46][47][48][49]. In general, cooperativity in substrate binding is revealed by a sigmoidal curve of the substrate-induced displacement of the spin equilibrium of the heme iron, which is known to be an important determinant of the catalytic efficiency and coupling of cytochromes P450 [50][51][52].…”

Section: Introductionmentioning

confidence: 99%

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

Fernando

Davydov

Chin

et al. 2007

Archives of Biochemistry and Biophysics

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The contribution of conformational heterogeneity to cooperativity in cytochrome P450 3A4 was investigated using the mutant L211F/D214E/F304W. Initial spectral studies revealed a loss of cooperativity of the 1-pyrenebutanol (1-PB) induced spin shift (S 50 = 5.4 μM, n = 1.0) but retained cooperativity of α-naphthoflavone binding. Continuous variation (Job's titration) experiments showed the existence of two pools of enzyme with different 1-PB binding characteristics. Monitoring of 1-PB binding by fluorescence resonance energy transfer from the substrate to the heme confirmed that the high affinity site (K D = 0.3 μM) is retained in at least some fraction of the enzyme, although cooperativity is masked. Removal of apoprotein on a second column increased the high spin content and restored cooperativity of 1-PB binding and of progesterone and testosterone 6β-hydroxylation. The loss of cooperativity in the mutant is, therefore, mediated by the interaction of holo-and apo-P450 in mixed oligomers.

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Pyrene·Pyrene Complexes at the Active Site of Cytochrome P450 3A4: Evidence for a Multiple Substrate Binding Site

Cited by 91 publications

References 19 publications

Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2

Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2

Spectral resolution of a second binding site for Nile Red on cytochrome P4503A4

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

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