Document Detail


Potent mechanism-based inactivation of cytochrome P450 2B4 by 9-ethynylphenanthrene: implications for allosteric modulation of cytochrome P450 catalysis.
MedLine Citation:
PMID:  23276288     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Haoming Zhang; Sean C Gay; Manish Shah; Maryam Foroozesh; Jiawang Liu; Yoichi Osawa; Qinghai Zhang; C David Stout; James R Halpert; Paul F Hollenberg
Related Documents :
18655058 - The crystal structures of the psychrophilic subtilisin s41 and the mesophilic subtilisi...
23224618 - Investigation of binding properties of umbelliferone (7hydroxycoumarin) to lysozyme.
18594878 - Statistical analysis of structural characteristics of protein ca2+-binding sites.
8475088 - Calmodulin-binding domain of recombinant erythrocyte beta-adducin.
1306308 - The il-2/il-2 receptor system: involvement of a novel receptor subunit, gamma chain, in...
833468 - The influence of fatty acids on the binding of bilirubin to albumin.
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2013-01-04
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-15     Completed Date:  2013-03-07     Revised Date:  2014-03-19    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  355-64     Citation Subset:  IM    
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Allosteric Regulation / drug effects
Animals
Aryl Hydrocarbon Hydroxylases / antagonists & inhibitors*,  chemistry,  metabolism*
Catalytic Domain
Crystallography, X-Ray
Kinetics
Molecular Docking Simulation
Phenanthrenes / pharmacology*
Protein Binding
Protein Conformation / drug effects
Rabbits
Grant Support
ID/Acronym/Agency:
AA020090/AA/NIAAA NIH HHS; CA016954/CA/NCI NIH HHS; ES003619/ES/NIEHS NIH HHS; GM077430/GM/NIGMS NIH HHS; GM098538/GM/NIGMS NIH HHS; R01 CA016954/CA/NCI NIH HHS; R01 ES003619/ES/NIEHS NIH HHS; R01 GM098538/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/9-ethynylphenanthrene; 0/Phenanthrenes; EC 1.14.14.1/Aryl Hydrocarbon Hydroxylases; EC 1.14.14.1/cytochrome P-450 CYP2B4 (rabbit)
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  A General Mechanism for the Copper- and Silver- Catalyzed Olefin Aziridination Reactions: Concomitan...
Next Document:  A New Mild Method for the C-Acylation of Ketone Enolates. A Convenient Synthesis of ?-Keto-Esters, -...