Transcript QM/MM Study of Cytochrome P450 BM3

QM/MM Study of Cytochrome
P450 BM3 Catalysis Mechanism
and Application in Drug Design
P450: Super family of monooxygenases
Function:
 metabolism of carbon-source organic molecules:
hormones, vitamin D etc.
 detoxification of xenobiotic compounds
 synthesis of biologically active compounds
Why interesting in drug design
The eve of ADME in High-Throughput Screening
Absorption by the intestine
Diffusion to the organism
Metabolism by the liver
Excretion by the kidney
& Toxicology
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One-half of potential drugs fail because of ADME/Tox
issues
Of the estimated $600 million cost of bringing a new
drug to market, more than $400 million of that is
wasted pursuing leads that turn out to be dogs.
P450 (CYP) enzymes--a class of enzymes responsible
for the metabolism of more than 50% of all known
drugs
Enzyme P450 BM3
RH + O2 +2e-+2H+
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R-OH+H2O
The function of P450 enzyme in drug
metabolism is responsible for the failure of a
good inhibitor to become a useful drug
P450 BM3: the closet bacterial analog to the
mammalian enzyme, soluble, three domains
(BMP heme domain, a FAD- and FMNcontaining NADPH-cytochrome P450
reductase)
Archives of Biochemistry and Biophysics 1999, 369(1), 24-29
Structure: heme Fe, S-Cys and H2O as ligands
Figure shows the porphyrin ring in the active site.
Putative conformational change
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Electron transfer:
NADPH->FAD->FMN
->BMP (heme)
Conformational change
facilitated by the “hinge”
moving between a “closed”
conformation and an “open”
conformation
FMN-heme distance:18Å->8Å
Dutton’s line: electron transfer
kET=k0ETexp[-R(t)]
Productive electron
transfer:
within 14–15 Å
Munro, Andrew et al Trends in
Biochemical Sciences 2002
Catalytic cycle of
cytochrome P450
6a, 6b, and 7: putative, no
experimental structure
RDS: 2nd e- injection
Intermediate compound I :
oxyferryl/porphyrin π-cation
radical
X-ray structure and product
1JPZ, X-ray 1.65 Å, BMP with
N-Palmitoylgolycine
3200 mol min-1(mol of enzyme)-1
(NADPH rate, 37°C), 50% conversion
D. C. Haines et al., Biochemistry, 2001
Computational modeling
Computational modeling proposed the
following binding structure, B:
Jovanovic et al, JACS (2005)
Solid State NMR Experiment
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2D solid-state NMR 15N13CO
SPECIFIC CP spectrum of
13COLeu; 15N-Gly, 15N-Phe labeled
cytochrome P450 BM-3 bound
with NPG at 0 (red) and -30 °C
(blue).
The L86-F87 pair exhibits a
pronounced shift as a function of
temperature. The L86-F87 pair is
in the binding pocket, and in fact
F87 plays a “gatekeeper” role in
that its bulky side chain must be
rotated in order to allow for
substrate binding.
QM/MM
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Combined Quantum mechanics/ Molecular mechanics
(QM/MM) is a hybrid technique to model enzymecatalyzed reactions.
A small reactive part of the system is treated
quantum mechanically (i.e., by an electronic structure
method); this allows the electronic rearrangements
involved in a chemical reaction, namely bond
breaking and making, to be modeled. The large nonreactive part is described by molecular mechanics,
and the two regions are able to interact.
The combination of the efficiency and speed of the
MM force field with the versatility and range of
applicability of the QM method allows reactions in
large systems to be studied.
Docking structure and QM/MM modeling
QM region of P450 BM3:
Docking structure:
Schrodinger, Inc. (2004)
Total charge= -3 Spin multiplicity= 4
MM region: protein, explicit water shell
Activation Barrier in QM/MM
Doublet, QM/MM, U-DFT, without zero-point energy correction
Search for P450 in PDB!
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www.rcsb.org
Search for P450 or P450 3A4
Check the hits:
What domain? Ligands? # of residues?
X-ray or NMR? Resolution?