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Faculty
 John
B. Schenkman
Professor of Cell Biology
jschenkm@neuron.uchc.edu
1. The research interests of Dr. Schenkman are mainly in the
area of the cytochrome P450-containing monooxygenase system. The role of
cytochrome b5 as an enhancer of the monooxygenase reaction
and its site of interaction on P450 is under investigation. Studies in
this laboratory involve determination of the nature of interaction
between the proteins of the monooxygenase, including cytochrome P450,
NADPH-cytochrome P450 reductase, and cytochrome b5.
Perturbants of the interaction, including alteration of conductivity of
the medium using different ionic strength and inclusion of polyols, as
well as pH variation have been used to determine the nature of forces
driving the interaction. The influence of these perturbants on electron
transfer between the proteins and the direction of electron transfer are
examined. Stopped flow spectroscopy of electron transfer reactions are
utilized as well as product identification in the processes.
Based upon studies in this laboratory the hypothesis was raised that
cytochrome b5 serves as an electron buffer, taking an
electron from reduced cytochrome P450, then returning it after a second
electron reduces the cytochrome P450 and oxygen binds to it (See sketch
at left). The prosthetic group of hemoproteins is a relatively small 616
Da, compared with the mass of the native cytochrome b5 (17
kDa) or cytochrome P450 (50 kDa) (see below). Consequently, a mechanism
for precise alignment of the redox centers of the two proteins would be
needed for increased efficiency of electron transfer. The interaction
between cytochrome P450 and cytochrome b5 has been shown to
be by complementary charge-pairing involving conserved acidic residues
of cytochrome b 5. This interaction generally stimulates the
monooxygenase turnover of substrates. The factors responsible for the
enhanced turnover are of special interest. Dr. Schenkman is interested
in the topology of interaction between NADPH-cytochrome P450 reductase
and cytochrome P450 and between cytochrome b5 and cytochrome
P450. A goal is to try to try to track the route of electron transfer
from the surface of the cytochrome P450 to its buried heme prosthetic
group. Dr. Schenkman has recently published a review on cytochrome b5
:J.B. Schenkman and I Jansson, The many roles of cytochrome b5,
Pharmacology & Therapeutics 97:139-152 (2003),
http://p450.uchc.edu/papers.html.
2. Cytochrome b5 is an acidic protein. 25 of its
131 amino acid residues is either an aspartic acid or glutamic acid
residue. A number of these acidic residues are located around a cleft in
the protein in which the heme prosthetic group resides. Figure 1 shows
the crystal structure of this protein. Several of the acidic residues
around the heme edge, plus one of the heme propionate residues, has been
shown to participate in complementary charge-pairing with redox partners
of cytochrome b5. The structure of cytochrome P450cam
(CYP101) is known, and the heme is located under a shallow depression on
the proximal surface of the protein. It is believed that all of the
forms of cytochrome P450 might have a similar structure. Above the heme
of CYP101 are a number of cationic amino acids which could serve as
potential charge-pairing residues for an acidic reduction/oxidation
partner like cytochrome b5.
Modeling of putative interactions between potential charge-pairing
residues suggested the site of cyt b5 interaction on CYP101
is on the proximal surface over the heme. Competition of cyt b5
with putidaredoxin, the native electron transfer protein to CYP101, and
site directed mutagenesis of putative charge-pairing residues of both
proteins supported the model. Sequence alignments and structural
alignments of different mammalian forms of cytochrome P450 with CYP101
have been made, and putative residues that might charge-pair with
cytochrome b5 acidic residues of mammalian are being
determined. Figure 2a and b show the surface of CYP101 proximal to the
heme. We have shown that cytochrome b5 binding is at a site
over the heme of P450 (light blue or cyan color on right),
charge-pairing with cationic (dark blue) amino acid residues. Our
studies have made use of quartz crystal microbalance measurements of
cytochrome P450 binding to solid supports (Figure 3) and the binding of
cytochrome b5 to the P450. We have also used atomic force
microscopy to view molecules of cytochrome P450 binding to lipid
surfaces. These findings were reported as, Schenkman, et al.,
'Charge-dependent sidedness of cytochrome P450 forms studied by quartz
crystal microbalance and atomic force microscopy.' Archives of
Biochemistry and Biophysics 385:78-87, 2001.
3. In other studies, Dr. Schenkman and his group have noted
that a number of forms of cytochrome P450 are found in the developing
embryo and fetus (Choudhary, et al., Comparative expression profiling of
40 mouse cytochrome P450 genes in embryonic and adult tissues." Archives
of Biochemistry and Biophysics 414:91-100 2003), and have hypothesized
that these forms are involved in normal development ("Involvement of
cytochrome P450 in development", Proc. Indian Natn. Sci Acad. B69 (6):
939-941, 2003) http://p450.uchc.edu/papers.html . We have developed
heterologous expression systems using Escherichia coli for production of
different cytochrome P450 forms in order to determine their properties
and their possible involvement in a number of genetic disorders in
humans. The absence of a functional form of cytochrome 450 1B1 (CYP1B1)
has beenlinked to a disease phenotyope, Primary Congenital Glaucoma (PCG)
in humans. We have prepared this hemoprotein and studies its
characteristics and believe there is a role for this enzyme in normal
eye development. The PCG phenotype is characterized as improper
development of the trabecular meshwork, which serves as a filter for
fluids of the anterior chamber of the eye. The improper development
results in poor drainage of this chamber and pressure builds up that if
not aleviated damages the retina and results in blindness. Mice have an
orthologous form of this hemoprotein, Cyp1b1, and the Cyp1b1 -/-
(knockout) mouse has similar defects in the trabecular meshwork. We
examined two mutations of CYP1B1, where incomplete penetrance in the PCG
phenotype is seen. Each of these forms of cytochrome P450 had a single
point mutation that altered only one amino acid; G61E and R469W. In the
former a neutral amino acid, glycine, at position 61 is converted to an
acidic amino acid, glutamic acid. In the other mutation a basic amino
acid, arginine is converted to a hydrophobic amino acid, tryptophan. We
were able to show that the resultant forms of cytochrome P450 were
expressed, but had considerably lower activity than the wild type
enzyme. It was suggested that a possible reason for the incomplete
penetrance with these forms was the influence of environmental factors
that influenced the expression levels of the proteins, elevations which
enabled the individual to have a normal eye development. Jansson, et
al., "Effect of two mutations of humamn CYP1B1, G61E and R469W, on
stability and endogenous steroid substrate metabolism." Pharmacogenetics
11:1-9, 2001.
http://p450.uchc.edu/papers.html
4. Currently, we are studying several forms of cytochrome P450
that have orthologs in other mammalian and vertebrate species for a
possible role in psoriasis. At least two forms are known to be expressed
in epithelial cells, and one form, CYP2S1 has been found in psoriatic
skin to be elevated to levels considerably higher than adjacent,
uninvolved skin. We believe it is possible that altered expression of
certain forms of cytochrome P450 could cause enhanced growth and
development or regions of the skin, as well as influence the
inflammatory processes seen with this disease.
The Schenkman Lab home page
Publications
Selected Publications:
Sarfarazi M, Stoilov I, Schenkman JB. Genetics and biochemistry of
primary congenital glaucoma. Ophthalmol Clin North Am. 2003
Dec;16(4):543-54, vi. Review.
Choudhary D, Jansson I, Schenkman JB, Sarfarazi M, Stoilov I.
Comparative expression profiling of 40 mouse cytochrome P450 genes in
embryonic and adult tissues. Arch Biochem Biophys. 2003 Jun
1;414(1):91-100.
Estavillo C, Lu Z, Jansson I, Schenkman JB, Rusling JF. Epoxidation
of styrene by human cyt P450 1A2 by thin film electrolysis and peroxide
activation compared to solution reactions. Biophys Chem. 2003 May
1;104(1):291-6.
Zhou L, Yang J, Estavillo C, Stuart JD, Schenkman JB, Rusling JF.
Toxicity screening by electrochemical detection of DNA damage by
metabolites generated in situ in ultrathin DNA-enzyme films. J Am Chem
Soc. 2003 Feb 5;125(5):1431-6.
Schenkman JB, Jansson I. The many roles of cytochrome b5.
Pharmacol Ther. 2003 Feb;97(2):139-52. Review.
Munge B, Estavillo C, Schenkman JB, Rusling JF. Optimization of
electrochemical and peroxide-driven oxidation of styrene with ultrathin
polyion films containing cytochrome P450cam and myoglobin. Chembiochem.
2003 Jan 3;4(1):82-9.
Jansson I, Stoilov I, Sarfarazi M, Schenkman JB. Effect of two
mutations of human CYP1B1, G61E and R469W, on stability and endogenous
steroid substrate metabolism. Pharmacogenetics. 2001 Dec;11(9):793-801.
Schenkman JB, Jansson I, Lvov Y, Rusling JF, Boussaad S, Tao NJ.
Charge-dependent sidedness of cytochrome P450 forms studied by quartz
crystal microbalance and atomic force microscopy. Arch Biochem Biophys.
2001 Jan 1;385(1):78-87.
Stoilov I, Jansson I, Sarfarazi M, Schenkman JB. Roles of cytochrome
p450 in development. Drug Metabol Drug Interact. 2001;18(1):33-55.
Review.
Jansson I, Stoilov I, Sarfarazi M, Schenkman JB. Enhanced expression
of CYP1B1 in Escherichia coli. Toxicology. 2000 Apr 3;144(1-3):211-9.
Lu Z, Lvov Y, Jansson I I, Schenkman JB, Rusling JF. Electroactive
Films of Alternately Layered Polycations and Iron-Sulfur Protein
Putidaredoxin on Gold. J Colloid Interface Sci. 2000 Apr
1;224(1):162-168.
Rusling JF, Zhou L, Munge B, Yang J, Estavillo C, Schenkman JB.
Applications of polyion films containing biomolecules to sensing
toxicity. Faraday Discuss. 2000;(116):77-87; discussion 171-90. |
