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Drug Biotransormation
Glutathione transferases (GSTs) and related enzymes
66
Pharmacology
Graduate
11/04/2010

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Term
Discovery of GSTs happened in the 1880's when dogs were given bromobenzene and excreted a ______-containing metabolite called ______.
Definition
Sulfur-containing
mercapturic acid
Term
The mercapturic acid excreted in urine from dogs given bromobenzene was shown to be the ____________ derivative of bromobenzene.
Definition
N-acetyl-cysteine
Term
What is the name of the tripeptide that is the source of cysteine in the N-acety-cysteine derivative (mercapturic acid) of bromobenzene, etc.?
Definition
gamma-glutamylcysteinylglycine named glutathione
Term
__________ substances are common substrates for glutathione transferases (GSTs)
Definition
Electrophilic
Term
Describe the GST (mercapturic) pathway of bromobenzene.
Definition
Bromobenzme + CYP = bromobenzene with an oxirane ring

bromobenzene(w/ oxirane ring) + GST + GSH = glutathione conjugate(bromobenzne with OH and gamma-glutamylcysteinylglycine).... -H2O to restore aromatic ring and loss of OH group

bromobenzene glutathione conjugate + gamma glutamyl transpeptidase = bromobenzene cysteinyl-glycine conjugate

bromobenzene cysteinyl-glycine conjugate + cysteinyl-glycinase = cysteine conjugate

cysteine conjugate + N-acetyltransferase = N-acetylcysteine conjugate (Mercapturic Acid!)
Term
What is the cosubstrate for GST?
Definition
GSH (gamma-glutamylcysteinylglycine)
Term
Is GSH available in high concentrations in the body? At what concentration in most tissues? The eye? Liver?
Definition
Yes, in the liver 5mM.

The lens of the eye has the most (10mM)

Most tissues >0.2 mM
Term
Why do cells generally contain ample amounts of GSH?
Definition
Used as a cellular antioxidant
Term
What affects the cellular concentration of GSH?
Definition
The protein nutritional status of the cell (source of amino acid components)

Exposure to xenobiotics
Term
How is the concentration of GSH controlled in the cell?
Definition
By a negative feedback loop with the [GSH] inhibiting the gamma-glutamylcysteine synthetase
Term
What is the role of buthionine sulfoximine on the concentration of cellular GSH?
Definition
Buthionine sulfoximine inhibits gamma-glutamylcusteine synthetase (reduces [GSH])
Term
What role does diethyl maleate have on cellular [GSH]?
Definition
Diethyl maleate is a substrate for glutation conjugation and will lower the concentration of GSH in the cell by forming a GSH conjugate.
Term
How do oxidizing agents such as glutathione disulfide affect cellular GSH?
Definition
During futile cycling of thiocarbamides and 2-mercaptoimidazole by FMO, GSH can be converted to glutathione disulfide GSSG
Term
Give the equation for reduction of GSSG

How is it catalyzed? Does it happen readily?
Definition
GSSG + NADPH + H+ ---> 2GSH + NADP+

Catalyzed by glutathione reductase
Happens readily in cells (with NADPH present)
Term
What are common substrates for the GST enzyme superfamily?
Definition
Electrophiles:

aryl halides, aklyl halides, epoxides, alpha-beta unsaturated ketones, aliphatic sulfate esters
Term
How do GST's affect the toxicity of drugs?
Definition
GSH conjugates are generally detoxication products.
Term
Does GST act as an isomerase?
Definition
Yes, for some substrates

(also, GSH is used, but not consumed)
Term
What is the reaction of GST (w GSH) with aklyl halides like dibromoethane?
Definition
Formation of an episulfonium ion which is highly reactive and fan form an ethyl-glutathione aduct of a DNA base

NOT a detoxification pathway of GST
Term
What happens when alpha-beta unsaturated ketones react with GST? How is it facilitated?
Definition
A Michael-type 1,4-addition of GS

Facilitated by a Tyrosine in N-term of GST that H-bonds with GSH to make it more reactive

Tyrosine/Serine in Substrate binding site that H-bonds to oxygen in ketone to stabilize it.
Term
What happens to sulfate esters that react with GST?
Definition
H2SO4 leaves and GS anchors to the R group
Term
What happen nitroglycerine reacts with GST?
Definition
Nitroglylcerine becomes a nitro alcohol and the GS binds to the NO2 leaving group... this transition state then reacts with GSH to form GSSG and HNO2
Term
Describe the reaction of steroid type molecules with GST.
Definition
GST acts as a steroid isomerase, where the location of a double-bond is changed. The [GSH] remains constant, as it is needed but not consumed in the reaction.
Term
Describe the reaction of Maleylacetoacetate and GST?
Definition
GST acts as a Maleylacetoacetate Isomerase and converts 4-Maleylacetoacetate (CIS) to 4-Fumarylacetoacetate (TRANS)...

[GSH] remains constant, as it is needed for the reaction to occur, but is not consumed
Term
What happens with dichloroacetate reacts with GST?
Definition
Dichloroacetate gets converted to glyoxylate (via GST(z)!) a reaction that does not consume GSH, but requires it in the RxN.

The Glyoxylate product and the chlroo-glutathione intermediate can form adducts to residues in the GST(zeta) protein
Term
Describe the structure of GST
Definition
Exists as a dimer (generally homo), with each monomeric subunig weighting ~22,000-29000(ligandin).

Has one GSH binding site (G-site) and one substrate binding site(H-site).
Term
What is ligandin?
Definition
When the most abundant protein of GST was discovered, it was shown to bind a number of lipophilic organic molecules (azo-dye carcinogens, bilirubin, and polyciclic aromatic hydrocarbons)

GSTA1-1 (alpha class of GST) is the most abundant form of GST (about 5% of the liver cytosolic protein)
Term
What is s-aryl-transferase?
Definition
Another name for GST (GSTA1-1)/ligandin

Dimer, monomeric weight 22000-29000, 5% cytoslic liver protein, most abundant GST

name no longer used
Term
What is the most abundant GST in liver?
Definition
GST A1-1, this protin is about 5% of the liver cytosolic protein
Term
Which enzyme is responsible for making GSH conjugates?
Definition
GST
Term
Which enzyme is responsible for the isomerization of double bonds (specifically in sterols)
Definition
GST
Term
Describe the "ligandin" function of GSTs. Is it reversible?
Definition
The "ligandin function" is important for intracelular transport of organic molecules (including potentially toxic compounds)

Yes, this binding is reversible
Term
In what case would GST bind irriversibly to an electrophile?
Definition
If there's insufficient amounts of GSH in the cell, the electrophile will covalently bond to cysteine, serine, or threonine groups in the GST protein.
Term
To what three amino acids in GST would an elctrophile bind in the case of insufficient GSH in the cell?
Definition
Cysteine
Serine
Threonine
Term
In which fraction of the cell were GST's first discovered?
Definition
Cytosolic fraction, (however, there is now evidence for microsomal and mitochondrial forms)
Term
Describe the nomenclature of GST
Definition
Families/class - Roman capital letters (corresponding to Greek letters)
A alpha
M mu
P phi
T theta
Z zeta
S sigma
O omega
K kappa

Subfamilies - Arabic numbers (homodimers = same ; heterodimers = different)
Term
Historically, if two GST forms showed cross reactivity to the same antibody, they were considered to be in the same _________.

Today, with the advent of proteomics, a limit of _____% sequence identity. However, most GST's are >___% identical. (For humans, this puts GST's of the same class on the same chromosome.
Definition
Family/class
50%
70%
Term
Most forms of GST (except ____ and _____) use CDNB as a ______.
Definition
Zeta
Omega
Substrate
Term
In which three families of GST's are there considerable overlap, making them less selective enzymes?
Definition
Alpha, Mu, Pi

(all have conserved tyrosine residue in GSH-binding site)
Term
Describe the importance of the G-site in GST.
Definition
The G-site is the GSH binding site of GST. It binds and facilitates the formation of the GS- anion and ehances catalysis by lowering pKa.

GSH pKa 9.5
GST-bound GS- pKa 7.5
Term
What do alpha, mu, and pi GST's have in common?
Definition
A conserved tyrosine residue in the GSH-binding site

low selectivity
Term
What to theta and zeta classes of GST's have in common?
Definition
A conserved serine residue in the GSH binding site, activate thiol gp.
Term
The alpha, mu, and pi classes of GST's are readily inducible by chemicals... name inducers of alpha and mu.
Definition
Alpha - induced by phenobarbital, 3-methylcholanthrene, and various antioxidants
Pi - induced by antioxidants
Term
What is the response element involved with induction of GST A by 3MC?
Definition
Dioxin Response Element (DRE) is the regulatory response element for 3MC induction of GST alpha
Term
What is the response element involved with antioxidants and pro-oxidants for Alpha and Pi classes of GST's?
Definition
ARE (antioxidant response element)
Term
Explain the significance of thiol modification of Keap1 cytoskeletal binding protein.
Definition
Modification of the cysteine residues on Keap1 allos Nrf2 to accumulate and enter the nucleus.

The Nrf2 then forms a heterodimer with Maf. The dimer binds to ARE and enhances transcription of GSTA4-4 and glutamate cysteine modulatory subunites (and thus synthesis of GSH).
Term
How does activation of the ARE lead to decrased amounts of 4-HNE in the cell?
Definition
Transactivation of the ARE-driven genes by the Nrf2-Maf dimer increases the production of GSTA4, glutamate cysteine ligase catalytic, and glutamate cysteine modulatory elements.

The elevation in the amounts of GSTA4-4 and GST allow increased metabolism of 4-HNE and its elimination from the cell via MRP.
Term
How is 4-HNE eliminated from the cell?
Definition
Metabolism using GSTA4-4/GSH and elimination via MRP
Term
How does phenobarbital affect GST?
Definition
First of all, leads to an increase in liver size

Induces GST A1-1 specifically (ligandin)

Studies showed that maximal induction with PB lead to ~13% GST A1-1 liver cytosolic protein.
Term
Are polymorphisms common in GSTs?
Definition
Yes
Numerous alleles have been identified for common GSTs
Term
Why don't some people express an active GST M1-1 allele that detoxifies carcinogens in cigarette smoke?
Definition
Homozygous for GST M1*0
Term
Why don't some people express activeGST T1-1 allele?
Definition
Homozygous for GST T1*0
Term
Why are GST P's overexpressed in certain cancers?
Definition
Multi Drug Resistance phenomenon

Expression of GST P in blood is suggested as a tumor marker
Term
Name two physiologically produced electrophiles that have been propsed as regulators of GST expression
Definition
hydroxynonenal and maleylacetoacetate
Term
Does age affect GST expression?
Definition
Yes, fetus has none essentially, increases with age until ~20 yrs and then starts to slowly decrease (although varies greatly between individuals)
Term
Describe the tissue distribution of GSTs?

Where are alpha, mu and pi forms mostly found?
Definition
Found mainly in liver
Widespread in tissues - (especially intestine & kidney)

Alpha - lung
Mu - lung
Pi - placenta
Term
How can calss A, M, and P GST's be isolated from liver cytosol?
Definition

Affinity chromatography:

 

Media: Glutathione-agarose or S-hexylthutathione coupled to epoxy-activated agarose

 

Add liver cytosol, GSTs will bind to medium, wash with buffer to remove all other proteins.

 

Wash with high concentration of GSH to remove GST's from medium.

 

Reverse phase HPLC onlarge pore columns, tetected by UV/fluorescence

Term
In HPLC analysis of GST enzymes, wildtype male and female rats both express the ______ gene the most (similar to ligandin gene in humans)... However, when GST Z form is knocked out, the ______ gene is massively induced in both male and female rats.
Definition
GST A3
GST M1
Term
In the mercapturic pathway:
Where does glutathione conugation occur? By what enzyme?
Definition
Liver, GST
Term
In the mercapturic pathway

Where does cleavage of gamma-glutamyl group occur? By what enzyme?
Definition
Bile/Kidney (not in liver)

gamma-glutamyl transpeptidase - high levels in the kidney on outside of cell membranes, also found in the cytosol bound to the membrane
Term
In the mercapturic pathway:
Where does cleavage of the glycine group occur? By what enzyme?
Definition
Hydrolysis of cysteinyl-glycine occurs in the kidney, intestine, and other sites (not liver)

Enzymes that catalyze this rxn:
Cysteinylglycinase
Aminopeptidase-M

(Both are membrane-associated zinc metalloproteinases)
Term
Which enzymes are zinc metalloproteinases?
Definition
Cysteinylglycinases and Aminopeptidase-M's (those that hydrolyze cysteinyl-glycine conjugates)
Term
In the mercapturic pathway:

Where does acetylation of cysteine occur? By what enzyme?
Definition
Kidney

Cystein S-conjugate N-aetyl transferase, requires Acetyl-coenzyme A as the acetyl donor

Product is a non-toxic, readily excreted mercapturic acid (N-acetylcysteine conjugate)
Term
Where does most deacetylation occur? What are the reactants and products of deacetylation?
Definition
The kidney - it has high deacetylase activity

Mercapturic acid can be deacetylated to reform cysteine conjugates
Term
Which enzyme is pyridoxal phosphate dependent?
Definition
Beta-lyases that form thiol compounds, pyruvate, and ammonia from cysteine
Term
How do GSH conjugates like mercapturic acid and cysteine conjugates transport in the body?
Definition
Several transporter proteins assist in transport.
Term
The liver is the main site of ________. But the _______ and ________ are important in further processing.
Definition
GSH conjugates
Kidney & Intestine
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