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Cytochrome P450 in:
THE METABOLIZER
MUHS SMART Team: Alex Borden, John Fuller, Daniel Kim, Alex Martinez, Joe Puchner, Nick Bell, Jud Bro, Sam Broadnax, Joel Gebhard, Nate Griffin, Christian Gummin, Andrew
Keuler, Daniel Moldenhauer, Tommy Sabatino, Randy Spaulding, Ryan Sung, Caden Ulschmid
Teachers: Keith Klestinski and David Vogt Mentors: James Kincaid, Ph.D. Piotr Mak, Ph.D. Kazik Czarnecki, Ph.D., Department of Chemistry, Marquette University
ABSTRACT
Why can’t Grandpa drink grapefruit juice with his Lipitor? Why is Tommy hypersensitive to
aspirin? The answers lie in a study of cytochrome P450s (CYP101), a family of enzymes that are
responsible for the transformation of vitamins, pharmaceuticals and other foreign chemicals
into soluble and readily excreted molecules. This goal is achieved primarily by hydroxylation
reactions, which occur in these molecules through a series of extremely fast sequential
reactions, called an enzymatic cycle (Figure 1). In order to better understand certain
intermediates in the cycle, the reaction must be stopped at given points. In a particular variant
of cytochrome P450s, the presence of an aspartic acid near the active site causes immediate
protonation of the peroxo group, making it impossible to stop the hydroxylation reaction.
However, in the mutant form, CYP101 D251N, the aspartic acid is replaced with an asparagine,
which blocks protonation on an atomic level. Scientists need to study these molecules and
characterize the molecular structures of the reaction intermediates in order to understand what
factors affect the process, such as mutation of particular protein sites and blockage by
interfering chemicals. The Marquette University High School SMART Team (Students Modeling A
Research Topic) modeled both the wild-type and the D251N mutant of P450cam using MSOE’s
3D printing technology.
RAMAN SPECTROSCOPY
THE ENZYMATIC CYCLE
(A)
INSTRUMENTAL SETUP
RH
ferric
resting substrate-free
state
ferric
substrate-bound
ROH
e-
ferrous
H2O2
Compound I
(π-cation radical)
O2
H+
“Biological Blowtorch”Where the oxidizing of
inert substances begins
oxy
H2O
e-
H+
RESULTS
Cytochrome P450s are important molecules that are involved in drug metabolism. This
metabolism is accomplished through the activation of the enzymatic cycle, which oxidizes
the foreign substances into more benign molecules. Sometimes, interactions between
Cytochrome P450s and foreign substances involve what is known as "mechanism-based
inhibition". Normally, substances enter the P450 substrate-binding site and activate the
cycle, which causes the reaction to occur, and breakdown the chemical. However, for some
particular substances, such as those in grapefruit, a VERY reactive intermediate is formed
during the enzymatic cycle which actually reacts with an active site amino acid residue. This
event causes a covalent bond to form which causes the substance that entered to be
permanently attached at the active site. Therefore, these substances "block" the active site
from being able to bind the natural substrate, so that the effective concentration of the
enzyme is lowered. The important consequence of this enzyme decline is that other
pharmaceutical compound are not being metabolized as fast and can build up to levels that
are dangerous. These drugs, which are normally metabolized through insertion of oxygen
to make them hydrophilic, are now not undergoing this biotransformation, leading to very
dangerous levels of these drug in the body. Just because someone takes the prescribed
dose of a drug does not protect the user from overdosing. It is for precisely this reason that
scientists are devoting time and research to better understand the function and stages of
these molecules.
ferric hydroperoxo
(B)
R-H
+
O2
2e ,. 2H +
P450
ferric peroxo
A. Oxy
Cytochrome P450cam
B. Hydroperoxo
Cytochrome P450cam mutant D251N
A. Oxy
B. Peroxo
v
R-OH
+
H2 O
Figure 1. The scheme of cytochrome P450 enzymatic cycle (A), the overall
hydroxylation reaction catalyzed by cytochromes P450 (B). The R-H means
substrate, the R-OH means hydroxylated substrate. The boxes indicate the
unstable intermediates, which can be mapped using Raman spectroscopy.
ν(O-O)
DRUG METABOLISM DEPENDS
ON CYTOCHROME P450
ν(O-O)
H+
C.
Hydroperoxo
The Active Site
Fig. 2A
Fig. 2B
Figure 3. The RR spectra and difference
traces of cytochrome P450cam in their oxy
(A) and hydroperoxo (B) forms.
Figure 4. The RR spectra and difference
traces of cytochrome P450cam in their oxy
(A), peroxo (B) and hydroperoxo (C) forms.
RAMAN SPECTROSCOPY
The chemistry behind the metabolism of drugs must be understood in order to locate
and prevent the problems caused by outside chemicals. Scientists use a technique
called resonance Raman spectroscopy, which measures movements of bonds through
wavelengths. This method allows us to see bonds such as (O-O) stretches of the
unstable intermediates, that are otherwise not accessible using other methods.
1dz8.pdb
Pictures courtesy of emedicalstate.com and dailyfitnessmagz.org
2a1n.pdb
The cycle is initiated by the binding of the substrate of a foreign chemical to the active site
of the Cytochrome P450. This causes a conversion of the heme group (gray) which makes
it susceptible to reduction. The reduction transforms the active site to the ferrous state,
which can then bind the dioxygen, forming an oxy-complex (green box, Fig. 1). The
delivery of another electron generates the peroxo-intermediate (blue box, Fig. 1), which is
then protonated to form the hydroperoxo intermediate (red box, Fig. 1). The key step
follows, in which delivery of another proton causes cleavage of the O-O bond so as to
generate the highly potent oxidizing species known as Compound I (orange box, Fig. 1); it
is this species which is capable of oxidizing even relatively inert substrates. In the mutant
being discussed in this poster, an acidic side chain (Asp251) (Fig. 2A) is replaced by an
amide (Gln251) (Fig. 2B), severely restricts protonation of the peroxo-intermediate,
allowing it to be trapped and structurally characterized by resonance Raman spectroscopy.
Made by the MSOE Center for Biomolecular Modeling using 3D Z-Corp printing.
A SMART Team project supported by the National Institutes of Health Science Education Partnership Award (NIH-SEPA 1R25RR022749) and an NIH CTSA Award (UL1RR031973).
Conclusion
By studying the enzymatic cycle of Cytochrome P450, scientists will be better able to
understand the complete functions of drug metabolism. In particular, scientists will
understand how Cytochrome P450 reacts with outside chemicals leading to health
implications and safer drug use. Specific parts of the cycle play specific roles in metabolism.
All these parts must be studied singularly in order to understand the complete function of the
protein. By understanding the particular aspects of the cycle, scientists and doctors will be
able to understand effects of drugs such as overdosing and incomplete metabolism, which can
lead to health concerns such as death.
Acknowledgement: Dr. Kincaid’s Group and the MUHS SMART would like to acknowledge Professor Stephen
Sligar and his group at the University of Illinois at Urbana-Champaign for their helpful collaboration.