Cytochrome P450 proteins in humans are drug

metabolizing enzymes and enzymes that are used to

make cholesterol, steroids and other important
lipids such as prostacyclins and thromboxane A2.
These last two are metabolites of arachidonic acid.
Mutations in cytochrome P450 genes or deficiencies
of the enzymes are responsible for several human
diseases. Induction of some P450s is a risk factor in
several cancers since these enzymes can convert
procarcinogens to carcinogens. P450 enzymes play
a major role in drug interactions.
Model of rat P450 2B1, showing mode of membrane attachment
CytP450Oxidase-CYP2C9
The CYP3A subfamily is one of the most
important drug metabolizing families in
humans. The crystal structure of 3A4 is known,
but confidential and heavily patented. CYP3A4
is "the most abundantly expressed P450 in
human liver". (Arch. Biochem. Biophys. 369,
11-23 1999). The color of perfused liver is due to
this protein. CYP3A4 is known to metabolize
more than 120 different drugs. Some of these
are well known and I give a list here of some of
the recognizable ones.
CYP3A4 Substrates
Acetominophen (Tylenol)
Codeine (narcotic)
Cyclosporin A (an immunosuppresant),
Diazepam (Valium)
Erythromycin (antibiotic)
Lidocaine (anaesthetic),
Lovastatin (HMGCoA reductase inhibitor,
a cholesterol lowering drug),
Taxol (cancer drug),
Warfarin (anticoagulant).
Poisoning by acetominophen overdose is caused by
P450 enzymes in the liver and kidney that convert
acetominophen into a very toxic intermediate that can
react with cellular macromolecules to damage cells and
eventually kill them. This intermediate normally reacts
with glutathione, a natural antioxidant in cells. It is
only when the glutathione is depleted that cell death
can occur. That's why acetominophen overdoses don't
have any serious symptoms until 3-4 days later. This
problem is worse in alcoholics, since they have induced
P450 enzymes that make more of the toxic
intermediate.
Fig. 1. (A) Overall fold of CYP3A4, colored from blue at the N terminus to green, to yellow, to red at the C
terminus

P. A. Williams et al., Science 305, 683 -686 (2004)

Published by AAAS
Fig. 2. Stereoimage showing the structural overlay of CYP3A4 in red, with P450 BM3 (molecule A of PDB
entry 1BU7) in blue, and P450 EryF (PDB entry 1OXA) in yellow

P. A. Williams et al., Science 305, 683 -686 (2004)

Published by AAAS
 Fig. 3. Stereoview of metyrapone bound to the heme of CYP3A4

P. A. Williams et al., Science 305, 683 -686 (2004)

Published by AAAS
 Fig. 4. Peripheral binding of progesterone to CYP3A4

P. A. Williams et al., Science 305, 683 -686 (2004)

Published by AAAS
Fig. 2.
 Fig. 2.
Overall structures of CYP3A4 in complex with ketoconazole (A)
and erythromycin (B). Structures are shown in dark gray with
color highlighting of helices F to G (residues 202–260) in red,
helix I (residues 291–323) in green, and the C-terminal loop
(residues 464–498) in blue. The complex structures are superimposed
on the ligand-free structure (Protein Data Bank ID code 1TQN) shown
in light colors. Orange arrows indicate the direction of coordinate
shifts in the F-G region relative the ligand-free structure.

Proc Natl Acad Sci U S A. 2006 September 12; 103(37): 13682–

13687. Published online 2006 September 5.
doi: 10.1073/pnas.0603236103.
Copyright © 2006 by The National Academy of Sciences of the USA
Aflatoxin biotransformation. Aflatoxin is produced by molds that
contaminate peanuts and grains. Cytochrome P450 adds an oxygen atom
(shown with an arrow), forming a reactive three-membered epoxide ring.
Ideally, other enzymes then add a glutathione molecule to the epoxide,
making it soluble and ready to be removed from the body. However, the
epoxide intermediate is highly reactive, attacking DNA bases. In the
lower figure, activated aflatoxin has bonded to a guanine ring in the
DNA helix, and the flat carbon-rich ring system (shown in white and
light gray) has inserted between the DNA bases. The extra bulk of the
molecule distorts the helix (notice how stretched the helix is top to
bottom) and leads to misreading of the genetic sequence when the DNA
is replicated. Coordinates were taken from entry 1hm1 at the Protein
Data Bank.
X-ray Crystal Structure of Horse Cytrocome c
Cytochrome c Heme Ligation and Covalent Attachments
to Cys14 and Cys17
Block diagram of CO flash photolysis apparatus
Effect of nifedipine, erythromycin, and quinidine on kinetics of CO
binding to P450 3A4. Progress curves in the absence (a) and in the
presence of nifedipine (b), erythromycin (c), and quinidine (d),
respectively. The CO concentration was 20 µM; substrate concentration
was 300 µM. P450 3A4 concentration: 0.5 nmol/ml in phosphate-
buffered saline (pH 7.1) containing 20 20% (w/v) glycerol; temperature,
23 °C.
Data Analysis: A kinetic difference method was applied to kinetically
distinguish the individual P450 species from the total P450 species. This
approach evaluates the difference between the kinetic profiles obtained in
the presence and the absence of a P450 effector and thus effectively
cancels out the contributions from P450s that do not bind the effector.
Using this approach, kinetic parameters for individual effector-specific
P450 3A4 (and 1A1) species were obtained by least squares fitting of the
difference curves to the kinetic difference Equation 1,

(Eq. 1)

where A’t and At are the absorbance changes observed at time t
for the reactions in the presence and the absence of effector. ai and
ai are the absorbance changes, and ki and ki are the pseudo-first
order rate constants for the effector-specific P450 in the presence
and the absence of effector, respectively. Thus, when a single P450
species is perturbed (n = 1), this equation simply reduces to a
difference of two exponential terms.
Difference kinetic analysis of the CO binding curves in Fig. 2.
Difference traces are shown for nifedipine (A), erythromycin (B), and
quinidine (C), as calculated from the differences between curves b and a,
c and a, and d and a in Fig. 2, respectively. The solidlines represent the
A, effect of BP and ANF on CO binding kinetics of P450 1A1. Progress curves in
the absence (a) and the presence of BP (b) and ANF (c). The CO concentration was
20 µM; effector concentration was 20 µM; P450 1A1 concentration was
0.3 nmol/ml in 0.1 M sodium phosphate buffer (pH 7.5) containing 20% (w/v)
glycerol. The temperature was 23 °C. B, difference between traces b and a in A for
BP. C, difference between traces c and a in A for ANF. The solid lines represent the
best fit according to the kinetic difference Equation 1.
Effect of BP and ANF on kinetics of CO binding to P450 3A4. A, progress curves in the
absence (a) and the presence of BP (b), ANF (c), and BP + ANF (d). The CO concentration was
20 µM; effector concentration was 20 µM. P450 3A4 concentration was 0.8 nmol/ml in
phosphate-buffered saline (pH 7.1) containing 20% (w/v) glycerol. The temperature was 23 °C.
Difference traces are shown for BP (B), ANF (C), and BP + ANF (D), as calculated from the
differences between curves b and a, curves c and a, and curves d and c in A, respectively. The
solid lines represent the best fit according to the kinetic difference Equation 1.
Relationship between BP hydroxylase activity and amount of ANF-bound P450 3A4
species II. Kinetic difference curves were obtained for different concentrations of ANF by
subtracting the CO binding curve obtained in the presence of ANF alone from that obtained for
BP + ANF to yield a difference curve similar to that in Fig. 2D. Difference analyses were then
performed to calculate a1, which reflects the amount of ANF-bound species II. CO binding
kinetics and activity measurements were performed in the absence (1) and the presence of
1 (2), 5 (3), and 20 µM ANF (4), and the corresponding a1 and BP hydroxylase values were
plotted. The respective a1 values were 0, 0.0041, 0.0087, and 0.0160, and the respective BP
hydroxylase activities were 1.8, 8.4, 44.5, and 82.1 pmol min nmol. P450 3A4 concentration
Schematic representation of P450 interactions with BP and ANF. The binding of a single P450
1A1 species with either BP or ANF is depicted on the left. The top of the right side shows binding of
BP to P450 3A4 species I (left column) and ANF binding to species II (right column). In addition, BP
binds to ANF-bound species II but not to ANF-free species II.
Thank You!