Ilia G. Denisov, Yelena V. Grinkova, Prithviraj Nandigrami, Mrinal S. Shekhar,
Emad Tajkhorshid, and Stephen G. Sligar.
Allosteric interactions in human cytochrome P450 CYP3A4: The role
of phenylalanine 213.
Biochemistry, 58:1411-1422, 2019.
(PMC: PMC7528618)
DENI2019-ET
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 in POPC Nanodiscs three mutants, F213A, F213S, and F213Y,
and
compared them with the wild-type CYP3A4 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 reduced activation of CBZ epoxidation
by PGS, most pronounced in F213A mutant. Using all-atom molecular
dynamics simulations we compared dynamics of WT CYP3A4
and the F213A mutant incorporated in the lipid bilayer and the
effect of the presence of PGS molecule at the allosteric peripheral
site and evaluated the critical role of Phe213 in mediating the
heterotropic allosteric interactions in CYP3A4.
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