SL22105-Lec LV-5 Glutathione -S transferases and conjugation

Lecture LV5: Glutathione-S-Transferases And Glutathione Conjugation

Detoxifies electrophilic drugs and toxic metabolites

By reacting with electrophile e.g. UDP-glucuonic acid with nucleophiles

Structure of glutathione

Disulphide bond results from oxidation

Purpose of glutathione

Reducing agent

protects against radicals, reactive oxygen species

What enzyme catalyses this reduction

NADPH-dependent reductase

Glutathione-S-Transferases super families

Cytosolic, microsomal, mitochondria enzymes

for drug metabolism

Cystolic vs microsomal structure

Cytosolic enzymes are dimers (2 sub-units).

Microsomal enzymes are trimers (3 sub-units) and mostly metabolise arachidonic acid (inflammatory mediators)

Reaction of Glutathione with Epoxides

Remember detoxifies electrophiles (electron deficiency)

• SH is deprotonated by enzyme

• Increases activity

• Protonates O which is a bad leaving group to form OH

• Important for metabolism of polycyclic aromatic compounds

Reaction of Glutathione by 1,4-Conjugate Addition

Double bond next to EWG (amide)

Conjugate addition (a.k.a. Michael addition) occurs with electron-deficient double bonds.

Requires electron-withdrawing (usually carbonyl) group.

Reaction proceeds via an enolate intermediate.

disulphide bond broken to increase reactivity

electrons move to area of low electron density, double bond

breaks double bond

breaks C=O

Form unstable O-

O- resonates

transfers electron

protonate double bond

Reaction of Glutathione in Paracetamol Metabolism

Reactive intermediate produced by cytochrome P₄₅₀ enzyme followed by dehydration.

Reaction with glutathione is a 1,4-conjugate addition.

overdosing on paracetamol

depletes glutathione

results in liver failure as it cannot process the oxidative species

Reaction Of Glutathione In 1,6-Conjugate Addition

Oxidation by cytochrome P₄₅₀ enzyme produces quinone intermediate.

Glutathione reacts with 1,6-conjugate addition.

Reaction restores aromaticity (shifts double bonds).

mercapturates

Conversion of Conjugates to Mercapturates

Processing occurs in the liver and kidneys, resulting in negatively charged mercapturate which is excreted in urine (or bile if Mw > 400 Da). The pathway involves sequential removal of glutamyl and glycyl residues, and an acetyl group is transferred from acetyl-CoA.

Conversion of Mercapturates by Cysteine Lyases

Cysteine lyases catalyze the conversion of mercapturates into cysteine derivatives, facilitating detoxification processes.

Conversion of Mercapturates by Cysteine Lyases

How do enzymes exist

As non-covalent dimers

Can be homo or hetero dimers with monomer subunits of 20-25 kDa

Subfamilies

20 isomers of GST creates subfamilies due to different combinations of monomers

e.g. alpha, mu, pi, sigma, tay, zeta, omega, kappa, GSTA1-1

Induction of GST enzymes

Induction of cytosolic and microsomal enzymes by physiological (oxidative) stress and xenobiotics (including drugs) can occur

What is specific and what is variable

Expression is tissue is specific

basal levels of enzymes are variable

Clinical implications of GST enzymes

Many drugs stimulate receptors that regulate fatty acid metabolism e.g. peroxisomal proliferation activating receptor

leads to higher rate of metabolism due to higher enzyme levels and activity

over expression of enzymes also associated with treatment resistance in cancers

Polymorphisms of GST

Occurs in the protein sequence but sometimes promoter region

reduces protein levels

effect of GST polymorphism

Polymorphisms can have difference levels of effect e.g., GST M1 polymorphisms are found in >20 different types of cancer, whilst polymorphisms in GSTA1 are only associated with colorectal cancer.

Decreased expression of GSTM1 due to polymorphisms is associated with decreased risk of prostate cancer.

Cytotoxics

Highly electrophilic

Polymorphisms of GST and cancer chemotherapy

Often uses cytotoxic drugs as therapeutic agents

these are usually electrophilic

ØCancer drugs often react with DNA but are also reactive with other nucleophiles.

ØMetabolism of these drugs is via the GST enzymes.

ØPolymorphisms in GSTA1 and P1 predict survival following chemotherapy – likely due different metabolic rates of the drugs.