Role of cytochrome B5 in modulating peroxide-supported cyp3a4 activity: evidence for a conformational transition and cytochrome P450 heterogeneity.
The role of cytochrome b(5) (b(5)) in the alpha-naphthoflavone (alpha-NF)-mediated inhibition of H(2)O(2)-supported 7-benzyloxyquinoline (7-BQ) debenzylation by heterologously expressed and purified cytochrome P450 3A4 (CYP3A4) was studied. Although alpha-NF showed negligible effect in an NADPH-dependent reconstituted system, inhibition of 7-BQ oxidation was observed in the H(2)O(2) system. Analysis of the effect of various constituents of a standard reconstituted system on H(2)O(2)-supported activity showed that b(5) alone resulted in a 2.5-fold increase in the k(cat) value and reversed the inhibitory effect of alpha-NF. In addition, titration with b(5) suggested that only 65% of the CYP3A4 participated in the interaction with b(5), consistent with cytochrome P450 (P450) heterogeneity. Study of the influence of b(5) on the kinetics of H(2)O(2)-dependent destruction of the P450 heme moiety suggested two distinct conformers of CYP3A4 with different sensitivity to heme loss. In the absence of b(5), 66% of the wild-type enzyme was bleached in the fast phase, whereas the addition of b(5) decreased the fraction of the fast phase to 16%. Finally, to locate amino acid residues that might influence b(5) action, several active site mutants were tested. Substitution of Ser-119, Ile-301, Ala-305, Ile-369, or Ala-370 with the larger Phe or Trp decreased or even abolished the activation by b(5). Ser-119 is in the B'-C loop, a predicted b(5)-P450 interaction site, and Ile-301 and Ala-305 are closest to the heme. In conclusion, the interaction of b(5) with P450 apparently leads to a conformational transition, which results in redistribution of the CYP3A4 pool.
Kumar
Santosh
S
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1031, USA. sakumar@utmb.edu
Davydov
Dmitri R
DR
Halpert
James R
JR
D000494:Allosteric Regulation; D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D001665:Binding Sites; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5 / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D020536:Enzyme Activators / Q000494:pharmacology*; D006418:Heme / Q000032:analysis / Q000378:metabolism; D006861:Hydrogen Peroxide; D066298:In Vitro Techniques; D007700:Kinetics; D010084:Oxidation-Reduction; D011804:Quinolines / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D001571:Benzoflavones; D020536:Enzyme Activators; D011804:Quinolines; D011994:Recombinant Proteins; C065956:7-benzyloxyquinoline; D006418:Heme; C011512:alpha-naphthoflavone; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human