QM/MM%20Study%20of%20Cytochrome%20P450%20BM3%20Catalysis%20Mechanism%20and%20Application%20in%20Drug%20Design

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QM/MM Study of Cytochrome P450 BM3 Catalysis
Mechanism and Application in Drug Design
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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

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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

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Enzyme P450 BM3
RH O2 2e-2H R-OHH2O

• 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 FMN-containing
  NADPH-cytochrome P450 reductase)

Archives of Biochemistry and Biophysics 1999,
369(1), 24-29
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Structure heme Fe, S-Cys and H2O as
ligands Figure shows the porphyrin ring in the
active site.
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Putative conformational change

• Electron transfer
• NADPH-gtFAD-gtFMN
• -gtBMP (heme)
• Conformational change
• facilitated by the hinge
• moving between a closed
• conformation and an open
• conformation
• FMN-heme distance18Å-gt8Å

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Duttons line electron transfer
kETk0ETexp-?R(t)

• Productive electron transfer
• within 1415 Å

Munro, Andrew et al Trends in Biochemical
Sciences 2002
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Catalytic cycle of cytochrome P450 6a, 6b, and
7 putative, no experimental structure RDS 2nd
e- injection Intermediate compound I
oxyferryl/porphyrin p-cation radical
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X-ray structure and product
3200 mol min-1(mol of enzyme)-1 (NADPH rate,
37C), 50 conversion D. C. Haines et al.,
Biochemistry, 2001
1JPZ, X-ray 1.65 Å, BMP with
N-Palmitoylgolycine
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Computational modeling
Computational modeling proposed the following
binding structure, B
Jovanovic et al, JACS (2005)
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Solid State NMR Experiment

• 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.

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QM/MM

• Combined Quantum mechanics/ Molecular mechanics
  (QM/MM) is a hybrid technique to model
  enzyme-catalyzed 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 non-reactive 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.

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Docking structure and QM/MM modeling
QM region of P450 BM3 Total charge -3 Spin
multiplicity 4 MM region protein, explicit
water shell
Docking structure Schrodinger,
Inc. (2004)
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Activation Barrier in QM/MM
Doublet, QM/MM, U-DFT, without zero-point energy
correction
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Search for P450 in PDB!

• www.rcsb.org
• Search for P450 or P450 3A4
• Check the hits
• What domain? Ligands? of residues?
• X-ray or NMR? Resolution?