3UAS

Cytochrome P450 2B4 covalently bound to the mechanism-based inactivator 9-ethynylphenanthrene

3UAS の概要

• エントリーDOI: 10.2210/pdb3uas/pdb
• 分子名称: Cytochrome P450 2B4, PROTOPORPHYRIN IX CONTAINING FE, phenanthren-9-ylacetaldehyde, ... (6 entities in total)
• 機能のキーワード: p450, cytochrome p450 2b4, monooxygenase, oxidoreductase, membrane protein, cyp 2b4, cyp lm2
• 由来する生物種: Oryctolagus cuniculus (European rabbit,Japanese white rabbit,domestic rabbit,rabbits)
• 細胞内の位置: Endoplasmic reticulum membrane; Peripheral membrane protein: P00178
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 55826.84

構造登録者

Gay, S.C.,Zhang, H.,Shah, M.B.,Stout, C.D.,Halpert, J.R.,Hollenberg, P.F. (登録日: 2011-10-21, 公開日: 2013-01-09, 最終更新日: 2024-11-27)

主引用文献

Zhang, H.,Gay, S.C.,Shah, M.,Foroozesh, M.,Liu, J.,Osawa, Y.,Zhang, Q.,Stout, C.D.,Halpert, J.R.,Hollenberg, P.F.
Potent Mechanism-Based Inactivation of Cytochrome P450 2B4 by 9-Ethynylphenanthrene: Implications for Allosteric Modulation of Cytochrome P450 Catalysis.
Biochemistry, 52:355-364, 2013

PubMed Abstract: The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and k(inact) are 0.2 and 0.25 min(-1), respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S(50) of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low-affinity site being the catalytic site and the high-affinity site on the protein periphery. Computer-aided docking and molecular dynamics simulations with one or two ligands bound revealed that the high-affinity site is situated at the entrance of a substrate access channel surrounded by the F' helix, β1-β2 loop, and β4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302.

PubMed: 23276288
DOI: 10.1021/bi301567z

実験手法

X-RAY DIFFRACTION (2.939 Å)