Detoxification in humans

1

• Detoxification in humans
• Phase I makes substrates more hydrophilic, less
  toxic, less activ
• Phase II makes substrates even more hydrophilic
  and ready for secretion
• (Phase III further metabolism of Phase II
  products)

2

• Detoxification in humans Phase I
• Hydrolysis esterases, proteases
• Oxidation cytochrome P450, FAD containing
  monooxygenases (oxidation at heteroatoms)
• Reduction cytochrome P450
• (Methylation SAM dependent methylases)

3

• Detoxification in humans Phase II
• Conjugation
• with glutathione by glutathion-S-transferase
• with glucuronat from UDPGA (UDP-glucuronic acid)
• with sulfate from PAPS (Phosphoadenylylsulfat)
• with acetyl group from acetyl CoA
• Methylation

4

• Cytochrome P450 undoubtedly the most popular
  research topic in biochemistry and molecular
  biology over the past half century
• It is probable that the cytochrome P450 system
  has been more extensively studied ... than any
  other enzyme or protein
• X-ray crystallographic determinations have had a
  major impact on our knowledge of P450
• David LV Lewis, 2001

5

• The cytochrome P450 family has
• Many members
• rice 450, humans 57, mouse 84,
  Chlamodymonas 10.
• Many functions
• Sex and drugs and alcohol modification of
  fatty acids, synthesis of steroids, degradation
  of hydrophobic substances (drugs), ethanol
  metabolism .

6

• The cytochrome P450 family
• Some of the catalyzed reactions (at least 20 more
  are known)

Halothane oxidation Halothane reduction Arginine
oxidation Cholesterol side-chain
cleavage Dehydrogenation Dehalogenation Azoreducti
on Deamination Desulphuration Amide
hydrolysis Ester hydrolysis Peroxidation Denitrati
on
Aromatic hydroxylation Aromatic
epoxidation Aliphatic hydroxylation Alkene
epoxidation N-dealkylation O-dealkylation S-dealky
lation N-oxidation N-hydroxylation S-oxidation Ald
ehyde oxidation Androgen aromatization
7

• The cytochrome P450 family
• The sequences are classified into clans (genes,
  that stem from a common anchestor) or clades
  (from organisms that stem from a common
  anchestor), classes (gt 20 identity 1,2, ...),
  families (gt 40 identity, eukaryotes 1, 2,
  3,... prokaryotes 101, 102, ...),subclasses (gt
  55 identity A, B, ...) and genes(1, 2, ...).

8

• The cytochrome P450 family and evolution

Plants and animals steroid synthesis
9

• The cytochrome P450 family and evolution

10

• P450 structure the cofactor heme is bound by a
  globin fold

Globin fold all helical, 3 3 helices
11

• Absorption of the heme group

The Soret peak at 450 nm is typical for P450
(pigment with 450 nm absorption). The exact
position of the peak depends on the ligands.
12

• Absorption of the heme group

Type I high spin iron (385-394 nm) Type II low
spin iron, direct iron ligation as in inhibitors
(416-420 nm, often shift to longer wavelengths,
CO 450 nm) Reverse type I (modified type II)
higher 420 nm peak, lower 390 nm peak
13

• Absorption of the heme group
• The spin state of the iron depends on the
  strength of the ligand field.

a1g
eg
b1g
eg
t2g
b2g
14

• P450 structure absorption of the heme group

Example Strong ligand field with CO
15

• Mechanism of action
• Activation of the dioxygen
• Structures of many substrate/oxygen complexes of
  P450cam (camphor hydroxylase from Pseudomonas
  putida) have been analyzed.
• The activated oxygen intermediate is created
  from dioxygen after two single electron reduction
  steps and cleavage of the oxygen-oxygen bond.

16

• P450 structure Activation of the dioxygen

17

• Mechanism of action
• Oxygen is bound in a bent end-on mode (remember
  naphthalene dioxygenase with non-heme iron
  side-on bound dioxygen).
• binding of oxygen pushes the camphor away only
  after dioxygen is reduced twice, camphor moves
  closer again. This prevents formation of reactive
  peroxides.
• the electrons for dioxygen reduction are
  provided by iron-sulfur proteins (bacterial and
  mitochondrial P450) or FAD/FMN-dependend
  NADPH-cytochrome P450 oxidoreductase (CPR,
  mammalian microsomal P450).

18

• Mechanism of action O2 reduction
• Iron-sulfur clusters and FMN are capable of
  single electron transfer steps.

In the P450 CPR complex, the electron moves
through the protein backbone.
19

• Drugs ethanol interaction
• Ethanol induces CYP2E and is oxidized by CYP2E.
  High ethanol concentrations can prevent other
  substrates to be degraded.

20

• QSAR quantitative structure-activity
  relationship
• The goal of QSAR is to predict binding affinities
  of new substrates for known enzymes based on the
  known (easy available) substrate properties.
• These properties include molecular weight, shape
  (length/width), HOMO-LUMO difference, dipole
  moment, number of hydrogen donors and acceptors,
  partition coefficient in octanol/water (logP),
  pKa, and many more.
• DGbind is usually correlated with (pseudo) energy
  terms
• number of H-bonds (each contributes a fixed
  amount)
• pKa (instead of Ka)
• logP (instead of P)

21

• QSAR example CYP2A6 subfamily

DG RT ln K (hier R 1.99 cal/molK, T 310 K)
22

• QSAR pros and cons
• Correlation can pinpoint important factors
• Potential substrates can be tested in silico
  (fast)
• Quality of the equation depends on the use of a
  representative subset
• Binding data must be available
• Binding mode might change upon substrate binding
• 3D details are difficult to correlate (needs lots
  of test compounds)