p450 - publications
Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of α-Naphthoflavone.
Biochem J. 2013 May 8;
Authors: Davydov DR, Davydova NY, Sineva EV, Kufareva I, Halpert JR
We investigated the relationship between oligomerization of cytochrome P450 3A4 (CYP3A4) and its response to alpha-naphthoflavone (ANF), a prototypical heterotropic activator. Addition of ANF resulted in over a two-fold increase in the rate of CYP3A4-dependent debenzylation of 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC) in human liver microsomes (HLM) but failed to produce activation in BD Supersomes™ or Baculosomes® containing recombinant CYP3A4 and NADPH-cytochrome P450 reductase (CPR). However, incorporation of purified CYP3A4 into Supersomes containing only recombinant CPR reproduced the behavior observed with HLM. The activation in this system was dependent on the surface density of the enzyme. While no activation was detectable at a lipid:P450 (L/P) ratio ≥ 750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization we probed P450-P450 interactions with a new technique based on luminescence resonance energy transfer (LRET). The amplitude of LRET in mixed oligomers of the heme protein labeled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes. Addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore, the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450-P450 interactions.
PMID: 23651100 [PubMed - as supplied by publisher]