09_P450

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Enzymatic detoxificationP450 enzymes

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Detoxification in humans

Phase I: makes substrates more hydrophilic, less toxic, less

activPhase II: makes substrates even more hydrophilic and

ready for secretion

(Phase III: further metabolism of Phase II products)

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Detoxification in humans: Phase I

Hydrolysis: esterases, proteases

Oxidation: cytochrome P450, FAD containingmonooxygenases (oxidation at heteroatoms)

Reduction: cytochrome P450

(Methylation: SAM dependent methylases)

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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

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Cytochrome P450 undoubtedly the most popularresearch topic in biochemistry and molecular biologyover the past half century

It is probable that the cytochrome P450 system has been

more extensively studied ... than any other enzyme orprotein

X-ray crystallographic determinations have had a major

impact on our knowledge of P450

David LV Lewis, 2001

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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 .

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The cytochrome P450 family:

Some of the catalyzed reactions (at least 20 more are known)

Aromatic hydroxylationAromatic epoxidationAliphatic hydroxylationAlkene epoxidationN-dealkylationO-dealkylation

S-dealkylationN-oxidationN-hydroxylationS-oxidationAldehyde oxidationAndrogen aromatization

Halothane oxidationHalothane reductionArginine oxidationCholesterol side-chain cleavageDehydrogenationDehalogenation

AzoreductionDeaminationDesulphurationAmide hydrolysisEster hydrolysisPeroxidation

Denitration

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The cytochrome P450 family:The sequences are classified into clans (genes, that stem from a

common anchestor) or clades (from organisms that stem from acommon anchestor), classes (> 20% identity: 1,2, ...), families (>40% identity, eukaryotes: 1, 2, 3,...; prokaryotes: 101, 102,

...),subclasses (> 55% identity: A, B, ...) and genes(1, 2, ...).

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The cytochrome P450 family and evolution

Plants and animalssteroid synthesis

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The cytochrome P450 family and evolution

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

Globin fold: all helical,~ 3 + 3 helices

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Absorption of the heme group

The Soret peak at 450 nm is

typical for P450 (pigment with450 nm absorption). The exactposition of the peak depends onthe ligands.

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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: 450nm)

Reverse type I (modified type II): higher 420 nm peak,

lower 390 nm peak

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The spin state of the iron depends on the strength

of the ligand field.

eg

t2g

a1g

b1g

eg

b2g

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P450 structure: absorption of the heme group

Example: Strong ligand field with CO

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Mechanism of action

Activation of the dioxygen

Structures of many substrate/oxygen complexes of

P450cam (camphor hydroxylase fromPseudomonas 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.

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P450 structure: Activation of the dioxygen

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Mechanism of action

Oxygen is bound in a bent end-on mode (remembernaphthalene dioxygenase with non-heme iron: side-on

bound dioxygen). binding of oxygen pushes the camphor away; only afterdioxygen is reduced twice, camphor moves closer again.This prevents formation of reactive peroxides.

the electrons for dioxygen reduction are provided byiron-sulfur proteins (bacterial and mitochondrial P450) orFAD/FMN-dependend NADPH-cytochrome P450oxidoreductase (CPR, mammalian microsomal P450).

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Mechanism of action: O2 reduction

Iron-sulfur clusters and FMN are capable of singleelectron transfer steps.

In the P450 CPRcomplex, the electronmoves through the

protein backbone.

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Drugs ethanol interaction

Ethanol induces CYP2E and is oxidized by CYP2E. Highethanol concentrations can prevent other substrates to be

degraded.

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QSAR: quantitative structure-activity relationship

The goal of QSAR is to predict binding affinities of newsubstrates for known enzymes based on the known (easyavailable) substrate properties.

These properties include molecular weight, shape(length/width), HOMO-LUMO difference, dipole moment,number of hydrogen donors and acceptors, partitioncoefficient 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)

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QSAR: example CYP2A6 subfamily

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

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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 arepresentative subset

Binding data must be available

Binding mode might change upon substrate binding

3D details are difficult to correlate (needs lots of testcompounds)

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09_P450