Allosteric Interactions in Human Cytochrome P450 CYP3A4

The Role of Phenylalanine 213

Ilia G. Denisov, Yelena V. Grinkova, Prithviraj Nandigrami, Mrinal Shekhar, Emad Tajkhorshid, Stephen Sligar

Research output: Contribution to journalArticle

Abstract

The role of Phe213 in the allosteric mechanism of human cytochrome P450 CYP3A4 was studied using a combination of progesterone (PGS) and carbamazepine (CBZ) as probe substrates. We expressed, purified, and incorporated into POPC Nanodiscs three mutants, F213A, F213S, and F213Y, and compared them with wild-type (WT) CYP3A4 by monitoring spectral titration, the rate of NADPH oxidation, and steady-state product turnover rates with pure substrates and substrate mixtures. All mutants demonstrated higher activity with CBZ, lower activity with PGS, and a reduced level of activation of CBZ epoxidation by PGS, which was most pronounced in the F213A mutant. Using all-atom molecular dynamics simulations, we compared the dynamics of WT CYP3A4 and the F213A mutant incorporated into the lipid bilayer and the effect of the presence of the PGS molecule at the allosteric peripheral site and evaluated the critical role of Phe213 in mediating the heterotropic allosteric interactions in CYP3A4.

Original languageEnglish (US)
Pages (from-to)1411-1422
Number of pages12
JournalBiochemistry
Volume58
Issue number10
DOIs
StatePublished - Mar 12 2019

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Cytochrome P-450 CYP3A
Phenylalanine
Cytochrome P-450 Enzyme System
Progesterone
Carbamazepine
Substrates
Allosteric Site
Lipid bilayers
Epoxidation
Lipid Bilayers
Molecular Dynamics Simulation
Titration
NADP
Molecular dynamics
Chemical activation
Atoms
Oxidation
Molecules
Monitoring
Computer simulation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Allosteric Interactions in Human Cytochrome P450 CYP3A4 : The Role of Phenylalanine 213. / Denisov, Ilia G.; Grinkova, Yelena V.; Nandigrami, Prithviraj; Shekhar, Mrinal; Tajkhorshid, Emad; Sligar, Stephen.

In: Biochemistry, Vol. 58, No. 10, 12.03.2019, p. 1411-1422.

Research output: Contribution to journalArticle

Denisov, Ilia G. ; Grinkova, Yelena V. ; Nandigrami, Prithviraj ; Shekhar, Mrinal ; Tajkhorshid, Emad ; Sligar, Stephen. / Allosteric Interactions in Human Cytochrome P450 CYP3A4 : The Role of Phenylalanine 213. In: Biochemistry. 2019 ; Vol. 58, No. 10. pp. 1411-1422.
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