Identification and Characterization of Cytochrome P4501A1 Amino Acid Residues Interacting with a Radiolabeled Photoaffinity Diazido-benzphetamine Analogue

Cvrk, Tomas; Hodek, Petr; Strobel, Henry W.

doi:10.1006/abbi.1996.0236

Cited by 14 publications

(5 citation statements)

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“…In a series of studies, Strobel and co‐workers (Shen and Strobel, 1995; Cvrk et al ., 1996) used peptide‐specific antibodies to map the putative CPR‐interacting domain of P4501A1 to a helical region spanning residues 269–281. Our results on the detailed mapping of binding sites are consistent with the general predictions of these studies.…”

Section: Discussionmentioning

confidence: 99%

Evolutionarily divergent electron donor proteins interact with P450MT2 through the same helical domain but different contact points

Anandatheerthavarada

Amuthan²,

Biswas³

et al. 2001

The EMBO Journal

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We have investigated the sites of N-terminally truncated cytochrome P4501A1 targeted to mitochondria (P450MT2) which interact with adrenodoxin (Adx), cytochrome P450 reductase (CPR) and bacterial flavodoxin (Fln). The binding site was mapped by a combination of in vitro mutagenesis, in vivo screening with a mammalian two-hybrid system, spectral analysis, reconstitution of enzyme activity and homology-based structural modeling. Our results show that part of an aqueous accessible helix (putative helix G, residues 264-279) interacts with all three electron donor proteins. Mutational studies revealed that Lys267 and Lys271 are crucial for binding to Adx, while Lys268 and Arg275 are important for binding to CPR and FLN: Additive effects of different electron donor proteins on enzyme activity and models on protein docking show that Adx and CPR bind in a non-overlapping manner to the same helical domain in P450MT2 at different angular orientations, while CPR and Fln compete for the same binding site. We demonstrate that evolutionarily divergent electron donor proteins interact with the same domain but subtly different contact points of P450MT2.

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Section: Discussionmentioning

confidence: 99%

Evolutionarily divergent electron donor proteins interact with P450MT2 through the same helical domain but different contact points

Anandatheerthavarada

Amuthan²,

Biswas³

et al. 2001

The EMBO Journal

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“…In 1996, Strobel and coworkers first reported the use of bifunctional crosslinking PAL [ 76 , 77 , 78 ]. They used a benzphetamine analog having two azido groups which were placed at opposite ends.…”

Section: Photo Affinity Ligands (Pals) As Binding Site Probesmentioning

confidence: 99%

“…They used a benzphetamine analog having two azido groups which were placed at opposite ends. Strobel and coworkers used radiolabeled p -azidocumene ( Figure 10 ) to identify the critical amino acid residue of P450 1A1 involved in binding of cumene hydroperoxide [ 76 ]. The experiments and studies performed by these and many other research groups have provided crucial data, which help in understanding the binding site of the CYP enzymes.…”

Section: Photo Affinity Ligands (Pals) As Binding Site Probesmentioning

confidence: 99%

Review of Ligand Specificity Factors for CYP1A Subfamily Enzymes from Molecular Modeling Studies Reported to-Date

et al. 2017

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The cytochrome P450 (CYP) family 1A enzymes, CYP1A1 and CYP1A2, are two of the most important enzymes implicated in the metabolism of endogenous and exogenous compounds through oxidation. These enzymes are also known to metabolize environmental procarcinogens into carcinogenic species, leading to the advent of several types of cancer. The development of selective inhibitors for these P450 enzymes, mitigating procarcinogenic oxidative effects, has been the focus of many studies in recent years. CYP1A1 is mainly found in extrahepatic tissues while CYP1A2 is the major CYP enzyme in human liver. Many molecules have been found to be metabolized by both of these enzymes, with varying rates and/or positions of oxidation. A complete understanding of the factors that govern the specificity and potency for the two CYP 1A enzymes is critical to the development of effective inhibitors. Computational molecular modeling tools have been used by several research groups to decipher the specificity and potency factors of the CYP1A1 and CYP1A2 substrates. In this review, we perform a thorough analysis of the computational studies that are ligand-based and protein-ligand complex-based to catalog the various factors that govern the specificity/potency toward these two enzymes.

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“…However, no PAL has been reported so far for CYP3A4, the major human liver P450 isoform. Several studies ( − ) have shown that peptides from CYP1A and 2B family isoforms can be covalently modified with different types of radiolabeled PALs, which are usually native or chemically modified substrates. In these studies, photolabeled peptides have been partially characterized and have been predicted by molecular modeling to line substrate-binding regions of these enzymes.…”

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confidence: 99%

Fluorescent Photoaffinity Labeling of Cytochrome P450 3A4 by Lapachenole: Identification of Modification Sites by Mass Spectrometry

et al. 2005

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While photoaffinity ligands (PALs) have been widely used to probe the structures of many receptors and transporters, their effective use in the study of membrane-bound cytochrome P450s is less established. Here, lapachenole has been used as an effective photoaffinity ligand of human P450 3A4, and mass spectrometry data demonstrating the efficient and specific photoaffinity labeling of CYP3A4 by this naturally occurring benzochromene compound is presented. Without photolysis, lapachenole is a substrate of CYP3A4 and can be metabolized to hydroxylated products by this enzyme. A high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) procedure was developed to analyze small amounts of intact purified CYP3A4, and analysis of the labeled protein showed the presence of one molecule of lapachenole bound per monomer of protein. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis after proteolytic digestion and isolation of fluorescent photolabeled peptides. Two peptide adducts accounting for >95% of the labeled peptides were isolated by HPLC, and both peptides, ECYSVFTNR (positions 97-105) and VLQNFSFKPCK (positions 459-469), were identified by nano-LC/ESI quadrupole time-of-flight (QTOF) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The sites of modification were further localized to positions Cys-98 and Cys-468 for each peptide by nano-LC/ESI QTOF tandem mass spectrometry (MS/MS). The results provided the first direct evidence for interaction between the PAL and the putative B-B' loop region, which may serve as a substrate access channel or as a part of the CYP3A4 active site. In conclusion, benzochromene analogues are effective PALs, which may be used in the study of other cytochrome P450 structures.

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Identification and Characterization of Cytochrome P4501A1 Amino Acid Residues Interacting with a Radiolabeled Photoaffinity Diazido-benzphetamine Analogue

Cited by 14 publications

References 0 publications

Evolutionarily divergent electron donor proteins interact with P450MT2 through the same helical domain but different contact points

Evolutionarily divergent electron donor proteins interact with P450MT2 through the same helical domain but different contact points

Review of Ligand Specificity Factors for CYP1A Subfamily Enzymes from Molecular Modeling Studies Reported to-Date

Fluorescent Photoaffinity Labeling of Cytochrome P450 3A4 by Lapachenole: Identification of Modification Sites by Mass Spectrometry

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