Selective suppression of the catalytic activity of cDNA-expressed cytochrome P4502B1 toward polycyclic hydrocarbons in the microsomal membrane: modification of this effect by specific amino acid substitutions.
Human hepatoma HEPG2 cells were infected with recombinant vaccinia virus vectors containing cDNAs encoding both known and variant rat cytochromes P450 (CYP). CYP2B1 and CYP2B2 cytochromes were equally well expressed (110-140 pmol/mg of microsomal protein) and catalyzed metabolism of 7,12-dimethylbenz[a]anthracene (DMBA). Their regioselectivity for DMBA metabolism paralleled that of the respective purified rat liver enzymes and reproduced previously reported regioselective differences between CYP2B1 and CYP2B2 [Wilson et al. (1984) Carcinogenesis 5, 1475-1483]. CYP2A1 and CYP2A2 expressed in HEPG2 microsomes exhibited nearly equal DMBA-metabolizing activities that closely matched that of purified CYP2A1. Although purified rat liver CYP2B1 was 3 times more active than purified rat liver CYP2B2, the expressed recombinant microsomal CYP2B1 (rCYP2B1) was 20 times less active than rCYP2B2, where activity matched that of the purified cytochrome. Microsomal suppression of rCYP2B1 catalytic activity was also observed for benzo[a]pyrene. Specific amino acid substitutions at equivalent positions of the completely homologous NH2-terminal halves of rCYP2B1 and rCYP2B2 changed this suppression effect. Thus, a L58----F, I114----F double mutant exhibited 3 times the normal activity for rCYP2B1 while remaining inhibitory for rCYP2B2. The single substitutions produced very different effects. The L58----F substitution prevented expression of rCYP2B1, while the I114----F substitution was inhibitory for both rCYP2B1 and rCYP2B2 (40 and 70%). A single E282----V mutation produced a stimulation of rCYP2B1 activity comparable to that of the L58----F, I114----F double substitution.(ABSTRACT TRUNCATED AT 250 WORDS)
Christou
M
M
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Mitchell
M J
MJ
Aoyama
T
T
Gelboin
H V
HV
Gonzalez
F J
FJ
Jefcoate
C R
CR
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000378:metabolism; D000596:Amino Acids / Q000235:genetics*; D001564:Benzo(a)pyrene / Q000378:metabolism; D002384:Catalysis; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics*; D005814:Genes, Viral; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology*; D009154:Mutation; D011083:Polycyclic Compounds / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000235:genetics
D000596:Amino Acids; D065607:Cytochrome P-450 Enzyme Inhibitors; D011083:Polycyclic Compounds; D011994:Recombinant Proteins; D001564:Benzo(a)pyrene; D013739:Testosterone; D015127:9,10-Dimethyl-1,2-benzanthracene; D004247:DNA; D003577:Cytochrome P-450 Enzyme System