Allosteric mechanisms in P450eryF probed with 1-pyrenebutanol, a novel fluorescent substrate

Davydov, Dmitri R.; Kumar, Santosh; Halpert, James R.

doi:10.1016/s0006-291x(02)00565-x

Cited by 32 publications

(78 citation statements)

References 19 publications

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“…Similar to results with CYP3A4 WT [48], this value is considerably lower than the S 50 determined by the spectrophotometric titration (5.4 ± 0.9 μM, see Table 1) and suggests a multi-site binding mechanism. As reported previously with P450eryF and CYP3A4 WT [14,40,48], the binding of 1-PB to the high-affinity binding site detected in our FRET experiments with CYP3A4 FEW does not result in the displacement of the spin equilibrium of the enzyme.…”

Section: Interactions Of Cyp3a4 With 1-pb Monitored By Fret To the Hesupporting

confidence: 89%

“…The series of absorbance and fluorescence spectra obtained in titration experiments were analyzed using principal component analysis (PCA) as described previously [14,40,58,59]. To interpret the spectral transitions in terms of the concentration of P450 species we used a leastsquares fitting of the spectra of principal components to the set of spectral standards of pure low-spin, high-spin and P420 species of the hemeprotein [49,58,59].…”

Section: Data Processingmentioning

confidence: 99%

“…In our previous studies we applied FRET from 1-PB to the P450 heme, in which fluorescencedue to determine the dissociation constant of the high-affinity binding site for this substrate in P450eryF and in CYP3A4 wild type [14,40,48]. The implementation of this approach used in the present work is based on a titration-by-dilution technique, where the concentration of the 1:1 mixture of the enzyme and substrate is changed gradually while keeping their molar ratio constant.…”

Section: Interactions Of Cyp3a4 With 1-pb Monitored By Fret To the Hementioning

confidence: 99%

“…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%

“…Recent use of fluorescence resonance energy transfer (FRET) from the fluorescent substrate 1-pyrenebutanol (1-PB) to the heme of cytochrome P450eryF (P450 107A1) [14,15,40] or CYP3A4 [48] has allowed us to determine the dissociation constant of the first binding event directly. The results suggest that both enzymes possess two binding sites with different affinities for 1-PB.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Interactions Of Cyp3a4 With 1-pb Monitored By Fret To the Hesupporting

confidence: 89%

Section: Data Processingmentioning

confidence: 99%

Section: Interactions Of Cyp3a4 With 1-pb Monitored By Fret To the Hementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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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Aluminum‐substituted heme domain of P450BM‐3 (BMP): Introducing a heme‐derived fluorescent probe for studies of substrate binding and protein–protein interactions in cytochromes P450

Davydov

Ponomarev

Bobrovnikova-Marjon

et al. 2013

Biotech and App Biochem

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We describe a novel technique for heme removal and replacement in the heme domain of P450BM-3 (BMP). The method was applied to obtain the aluminum-protoporphyrin IX (Al-PP) substituted derivative of BMP (Al-BMP). The overall yield of the purified Al-BMP was about 15% as related to the initial amount of the hemeprotein. Al-BMP possesses extensive fluorescence in the 550-650 nm region with excitation in the porphyrin absorbance bands. The protein was shown to bind substrates of P450BM-3 (palmitic, arachidonic, and cis-parinaric acids) with affinities similar to those of the native enzyme (3-6 μM). However, the substrate-induced changes in fluorescence of Al-PP reveal the existence of a second, low-affinity substrate-binding site, which cannot be detected by the spin shift in the native, heme-containing BMP. Using fluorescence resonance energy transfer, we have demonstrated that Al-BMP forms a complex with the flavoprotein domain of P450BM-3 labeled with 7-ethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin maleimide, revealing the affinity similar to that of native BMP (K d = 5 μM at 0.06 M ionic strength). Therefore, aluminum-substituted BMP may serve as a valuable tool in studies on the mechanisms of interactions of P450s with their substrates and protein partners.

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Substrate binding to cytochromes P450

2008

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P450s have attracted tremendous attention due not only to their involvement in the metabolism of drug molecules and endogenous substrates but also the unusual nature of the reaction they catalyze, namely the oxidation of unactivated C-H bonds. The binding of substrates to P450s, which is usually viewed as the first step in the catalytic cycle, has been studied extensively via a variety of biochemical and biophysical approaches. These studies were directed towards answering different questions related to P450s including, mechanism of oxidation, substrate properties, unusual substrate oxidation kinetics, function, and active site features. Some of the substrate binding studies extending over a period of more than forty years of dedicated work has been summarized in this review and categorized by the techniques employed in the binding studies.

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Allosteric mechanisms in P450eryF probed with 1-pyrenebutanol, a novel fluorescent substrate

Cited by 32 publications

References 19 publications

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

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

Aluminum‐substituted heme domain of P450BM‐3 (BMP): Introducing a heme‐derived fluorescent probe for studies of substrate binding and protein–protein interactions in cytochromes P450

Substrate binding to cytochromes P450

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