RT Journal Article
SR Electronic
T1 TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROME <em>B</em><sub>5</sub> AND OF SITE-DIRECTED MUTAGENESIS
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 155
OP 161
DO 10.1124/dmd.32.1.155
VO 32
IS 1
A1 Yamaguchi, Yoshitaka
A1 Khan, Kishore K.
A1 He, You Ai
A1 He, You Qun
A1 Halpert, James R.
YR 2004
UL http://dmd.aspetjournals.org/content/32/1/155.abstract
AB The active site topology of heterologously expressed CYP3A4 purified from an Escherichia coli expression system was examined using phenyldiazene. Incubation of CYP3A4 with phenyldiazene and subsequent oxidation yielded all four potential N-phenylprotoporphyrin IX regioisomers derived from attack on an available nitrogen atom in pyrrole rings B, A, C, or D (NB:NA:NC:ND = 6:73:7: 13). Further study using 28 active site mutants showed that substitution of residues closer to the heme, Ala-305, Thr-309, or Ala-370, with a larger residue caused the most drastic changes in regioisomer formation, which reflected the location of each amino acid residue replaced in a CYP3A4 homology model. Previous studies have suggested a conformational change in CYP3A4 upon binding of NADPH-cytochrome P450 reductase (CPR) or cytochrome b5 (b5). Therefore, regioisomer formation was also compared in the absence of redox partners and in the presence of CPR, b5, or both. Formation of all four regioisomers in CYP3A4 wild type, particularly the minor ones, was reduced in the presence of b5. CPR also greatly decreased the three minor isomers but increased the major isomer significantly. The presence of b5 and CPR restored minor isomer formation and suppressed the enhancement of NA formation caused by CPR alone. Interestingly, the effects of the redox partners differed among representative active site mutants. In particular, the increase in NC upon substitution of Ala-370 with Phe was significantly reversed in the presence of redox partners, strongly suggesting that a conformational change occurs around pyrrole ring C due to protein-protein interactions between CYP3A4 and CPR or b5. The American Society for Pharmacology and Experimental Therapeutics