The influence of vitamin B group on monooxygenase activity of cytochrome P450 3A4: pharmacokinetics and electro analysis of catalytic properties

Шумянцева, В. В.; Ших, Е. В.; Makhova, A.A.; Bylko, T V; Кукес, В. Г.; Sizova, Oksana S.; Раменская, Г. В.; Usanov, Sergey A.; Archakov, A. I.

doi:10.18097/pbmc20115703343

Cited by 5 publications

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“…Others have reported the immobilization of CYP3A4 on solid matrices such as gold electrodes allowing electrochemically driven drug metabolism, monolithic chromatographic supports in the development of immobilized enzyme reactors (IMERs), glass slides for analysis by total internal reflection microscopy (TIRFM), or silver nanoparticles in the development of a localized surface plasmon resonance (LSPR) spectroscopy-based biosensor . Various other surfaces have also been employed in the development of CYP3A4-based amperometric − and oxygen-sensing drug metabolism biosensors. However, in all the above cases, CYP3A4 was immobilized in a random manner, either covalently or noncovalently in biological membranes (microsomal or E. coli -derived) or in synthetic membranes such as Nanodiscs. , We report here what is, to our knowledge, the first functional immobilization of CYP3A4 in a covalent and oriented fashion, through site-specific modification of a single cysteine residue on its surface.…”

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confidence: 99%

Site-Specific Fluorescent Labeling and Oriented Immobilization of a Triple Mutant of CYP3A4 via C64

et al. 2012

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The generation of site-specific bioconjugates of proteins is highly desired for a number of biophysical and nanotechnological applications. To this end, many strategies have been developed that allow the specific modification of certain canonical amino acids and, more recently, noncanonical functional groups. P450 enzymes are heme-dependent monooxygenases involved in xenobiotic metabolism and in the biosynthesis of a variety of secondary metabolites. We became interested in the site-specific modification of these enzymes, CYP3A4 in particular, through our studies of their in vitro biocatalytic properties and our desire to exploit their remarkable ability to oxidize unactivated C-H bonds in a regio- and stereospecific manner. Obtained via a partial cysteine-depletion approach, a functional triple mutant of CYP3A4 (C98S/C239S/C468G) is reported here which is singly modified at C64 by maleimide-containing groups. While cysteine-labeling of the wild-type enzyme abolished >90% of its enzymatic activity, this mutant retained ≥75% of the activity of the unmodified wild-type enzyme with 9 of the 18 maleimides that were tested. These included both fluorescent and solid-supported maleimides. The loss of activity observed after labeling with some maleimides is attributed to direct enzyme inhibition rather than to steric effects. We also demonstrate the functional immobilization of this mutant on maleimide-functionalized agarose resin and silica microspheres.

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