Cytochrome P450
• Heme protein• Terminal oxidase of the mixed-function
oxidase (MFO) electron-transfer system• Located in the smooth endoplasmic
reticulum of all major organs and tissues• Uses NADPH as a source of reducing
equivalents• Inducible
Overall reaction
R-H + O2 + NADPH + H+
R-OH + H2O + NADP+
Ferric protoporphyrin IX
N
N-
N
-N
Fe+3
CH3 HCCH2
H3C
CH3
CH3
CH
CH2
CH2
H2CC
O
OH
H2C
H2CC
O
OH
Protoporphyrin IX
Catalytic cycle of cytochrome P450
from NADPH-cytC reductase
Fe3+
Fe3+-RH
Fe2+-RH
O2
[Fe2+-RH]
HO2-
[Fe2+-RH]
HO22-
Fe3+-RH
+ RH
+ e-
+O2
ROH
H+ + e-
NADPH
NADH
H2O
H2O2H+
H+
O2-.
P450 and reductase in endoplasmic reticulum
The P450 gene superfamily• Format of nomenclature:
CYPFamily/Subfamily/Gene• Family = 1, 2, …150 and counting
– ~40% aa similarity• Subfamily = A, B,…H…
– 55-65% aa similarity• Gene = 1, 2..10 or above
– >97% aa similarity (allelic variants)• Families grouped in Clans
Sub-Family family Gene
CYP1 A 1 BaP hydroxylation, O-deethyl’n(PAC-inducible 2 N-hydroxylation, O-deethylation
CYP2 A 1 Testosterone 7-hydroxylation2 Testosterone 15-hydroxylation
B 1 Aliphatic hydroxylation2 O-deethylation
C 1 - 20+2C19, mephenytoin hydroxylase
Demethylation
Deethylation
O
N
H2C CH3
H C CH3
O
OH
NH C CH3
O
+
Phenacetin Acetaminophen
C CH3
o
H
Acetaldehyde
OCH3
OH
COH
H
+
Anisole Phenol Formaldehyde
Sub-Family family Gene
CYP1 A 1 BaP hydroxylation, O-deethyl’n(PAC-inducible 2 N-hydroxylation, O-deethylation
CYP2 A 1 Testosterone 7-hydroxylation2 Testosterone 15-hydroxylation
B 1 Aliphatic hydroxylation2 O-deethylation
C 1 - 20+2C19, mephenytoin hydroxylase
Sub- Family family Gene
CYP2 D 1 - 6+2D6, debrisoquine hydroxylase
E 1 C- and N-hydroxylationsmall molecules
2 F 1
CYP3 A 1-43A4
CYP4 A 1 Lauric acid - and -1 hydroxylation
Sub-Family family GeneCYP11 (mito) A 1 Steroid 11-hydroxylation
CYP17 A 1 Steroid 17-hydroxylationCYP21 A 1 Steroid 21-hydroxylation
CYP51 A 1 (Plants, yeast)CYP52-66 A Yeasts, fungiCYP71-99, 701 PlantsCYP101 A 1 Pseudomonas putida P450cam
CYP102-132 A Bacteria
Induction
• Increased transcription• Increased protein synthesis• Enhanced stability of protein• Synthesis of enzyme with higher catalytic
activity
Example:Ah-locus mediated induction
AhR, receptor in cytoplasm, binds ligand: eg PAHs, TCDD, some PCBs
Bound AhR loses 2 heat-shock proteins (hsp90), becomes phosphorylated
Activated bound AhR migrates to nucleus, forms complex with Ah receptor nuclear translocation factor Arnt
AhR-Arnt complex binds to regulatory sequences in DNA (DRE, dioxin-responsive elements)
Transcription of CYP1A1 gene and other genes
Other “inducers” also interact with receptors
• CAR, responds to phenobarbital-type inducers, regulates CYP2B, CYP3A4, CYP reductase, transferases (?)
• PXR, CYP3A• PPARα, CYP4A• LXR, FXR control enzymes involved in
bile acid and lipid metabolism