WK09L2 - Biotransformation II (Ben) Flashcards by Leiel Lalo

What is the overall reaction performed by a CYP450 mono-oxygenase enzyme?

endo-/xenobiotic + O₂+ NADPH + H⁺

becomes

oxygenated endo-/xenobiotic + H₂O + NADP⁺

(one O of O2 goes to the biotic… one goes to water… both H atoms go to water)

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What are the 6 types of phase II conjugation reactions in biotransformation?

(in order of frequency/importance in the body)

Glucuronidation
Sulfation
Glutathione conjugation
Amino acid conjugation
Methylation
Acetylation / Acylation

GSGAMA

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What is the co-factor used in glucuronidation reactions?

What is it a derivative of and how is it made?

Other than biotransformation, what is it used for?

UDP-glucuronate

derivative of UDP-glucose
UDP-glucose is dehydrogenated leaving it with -COO^- on C6 of its ring (using 2 NAD⁺)
also used in formation of mucopolysaccharides

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What is the enzyme responsible for glucuronidation?

UDP-glucuronyl transferase (UGT)

a superfamily of enzymes broken into subfamilies 1 and 2

(subfamily 2 has further divisions into A and B categories)

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What are 3 examples of glucuronidated xenobiotics?

(w/ 3 examples of functional groups that can be glucuronidated)

Acetaminophen (on the phenolic -OH)
Ibuprofen (on a -COOH group)
p-Aminosalicylic Acid (on the amino group)

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What are the major substrates of the UGT1 and UGT2 isoenzymes?

UGT1: fenols, bilirubin
UGT2: steroids, bile acid

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What are some (6) inducers of UGT enzymes?

Morphine
Steroids
Bile Acids
Retionids
Polycyclic Aryl Hydrocarbons
Heterocyclic Aryl Hydrocarbons

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Where are UDP enzymes located?

What does this mean for their substrates and products?

How is this unique?

Embedded in the ER membrane with active sites in ER lumen

UDP-glucuronate must enter and glucuronide conjugates must leave the ER via transporters
is the only conjugation occuring in the ER lumen

(others are in the cytosol)

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What is the 2nd most important kind of conjugation?

Via what enzyme + where is it found?

Sulfation

addition of inorganic sulfate (to an -OH group or amine)
via sulfotransferase enzymes in the cytosol

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What is the co-factor which donates the inorganic sulfate in sulfation?

How is it made?

PAPS

(3’-Phosphoadenosine-5’-phosphosulfate)

made in 2 steps by PAPS synthetase using sulfate from Cys metabolism and 2 ATP

(enzyme is like a kinase, but transfers sulfate instead of phosphate)

creates an anhydride bond btwn sulfate + phosphate with high group transfer potential

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What are the general types of substrates for sulfation?

And a few specific examples?

General: phenols, alcohols, aromatic amines
Examples:
- estrone
- 3-OH-coumarin
- acetaminophen (not only glucuronidated!)
- methyl-DOPA

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What is the 3rd most important kind of conjugation?

Via what enzyme + where is it found?

Glutathione conjugation

via glutathione S-transferase enzymes

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Describe the structure of glutathione.

And its general functions?

Is a tripeptide made of Glu, Cys, and Gly with an unusual peptide bond btwn Glu-Cys at the γ-carboxyl (rather than α)

important endogenous water-soluble anti-oxidant
forms disulfide when oxidized by ROSs
involved in conjugation

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What is the molecular/energetic basis of glutathione conjugation?

(As in, what part of glutathione reacts with what part of the molecule to be conjugated?)

Reactivity of glutathione’s -SH group with an electrophilic reagent (R-X in img)

Results in thioether formation btwn the electrophile and glutathion.

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What happens in glutathione conjugation after glutathione is added to the endo-/xenobiotic in question?

Glu is hydrolyzed by a glutamyl transferase leaving only Cys and Gly
Then Gly is hydrolyzed by cysteinyl-glycinase leaving only Cys
And acetyl transferase now acetylates Cys amino grp leaving a mercapturic acid derivative

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What are the most importan endogenous substrates for glutathione conjugation?

Leukotrienes

several different leukotrienes are just the products of each step in the removal of the amino acid constituents of glutathione (when it is conjugated to arachidonic acid) mentioned in the previous card

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What is the 4th most important kind of conjugation?

Via what enzymes + where are they found?

Amino acid conjugation

via acyltransferases
found in mitochondria (at least the glycine transferase is)

What two AAs are most commonly used in amino acid conjugation?

Glycine

and

Taurine (made from Cys)

What is the unique first step in amino acid conjugation?

And an example of this with a molecule that is AA conjugated?

Activation of the molecule to be conjugated…

ex: carboxyl group of benzoic acid is bound to S-CoA using energy from one ATP via an acyl-CoA synthetase enzyme

After activation of the substrate molecule, what is the last step in amino acid conjugation?

The activated molecule is transferred to an amino acid using an N-acyltransferase enzyme

(Acyl-CoA glycinetransferase in the case of glycine conjugation)

In general, what are the substrates for amino acid conjugation?

And several specific examples?

General: Acyl-CoA derivatives of carboxylic acids
Examples:
- Benzoic Acid
- Salicylic Acid
- Nicotinic Acid
- Cinnamic Acid
- Bile Acids (cholic acid, deoxycholic acid)

What is the 5th most important kind of conjugation reaction?

What functional group can this conjugation be done to?

Via what enzymes + where are they found?

Acetylation of amine groups

via acetyltransferase enzymes either free in cytosol or ER-bound (with active sites in cytosol)

What are the general substrates for acetylation?

And some specific examples?

General: amides
Examples:
- sulfonamides
- clonazepam
- mescaline
- dapsone
- isonazid

What is the 6th most important kind of conjugation reaction?

What functional groups are conjugated in this way?

Via what enzymes + where are they located?

Methylation

usually phenolic -OH grps, sometimes amino groups

- via methyltransferase enzymes in cytosol

What is the common donor of methyl groups for methylation conjugations? How is it made?

**S-adenosyl methionine** - made by **methionine adenosyltransferase** using ATP and yielding PPi and Pi

What are the general substrates for methylation? And some specific examples?

* _General_: **catecholamines, phenols and amines** * _Examples_: * **Dopamine** * **Epinephrine** * **Pyridine** * **Histamine** * **Thiouracil**

Give an example of how the location of a methylation on a molecule can have drastic differences on the activity of the product.

With **norepinephrine**... * Methylation of the phenolic -OH (by COMT) leads to an inactive product * Methylation of the amine (by PMNT) leads to **epinephrine** formation

Conjugation products can be _deconjugated_ resulting in re-creation of an active intermediate... Give an example of this in the body.

* **Estrone** is created from **androstenedione** by **aromatase** in the uterus (and other tissues) * It is then _sulfated_ by sulfotransferases in the liver to _estrone-sulphate_ * Some breast tumors contain high amounts of **steroid sulfatase** enzymes which desulfate estrone-sulphate, re-activating it. * (Estrone then acts as a growth factor for these tumors.)

What are the 2 routes for elimination of conjugated molecules from the liver?

* **Blood** --\> filtration in the kidney --\> excretion via urine * **Bile** --\> secretion into the GI tract --\> excretion via feces

Describe the entry and exit of endo-/xenobiotics into and out of hepatocytes in the process of biotransformation.

* _Entry_ - usually **passive** transport into the hepatocyte via membrane transporters (uptake transporters = acronyms on left of img) * _Exit_ - almost always **active** transport, transporters have specificity for certain structural features to determine excretion to bile or blood (acronyms on right)

What are some of the transporters responsible for secretion of conjugated molecules from hepatocytes to _blood_? To _bile_?

* _Blood_ - **MRP1/3/6** (multi-drug resistance protein) * _Bile_ * __**MRP2** * **MDR1/3** (also multi-drug resistance protein) * **BSEP** (Bile Salt Export Pump)

What class of endo-/xenobiotic transport molecules used for removal of molecules from cells uses ATP? Describe them + their function.

**ABC transporters** (ABC = ATP-binding cassette) * 12 TM helices * 2 nucleotide binding domains * Some export **conjugated** molecules from the **cytosol**, others export **unconjugated toxins** from _within the membrane itself_ as a protective mechanism.

Describe how bile acid synthesis is a classic example of biotransformation.

* Cholesterol is an _endobiotic_ which can not be easily broken down. * Instead it is _hydroxylated_ by several CYP450 enzymes in the liver (first = 7-α hydroxylase) to form **cholate** * Then cholate is _conjugated_ to form **glycocholate**

Describe the _1st phase_ biotransformation of heme.

* It is first oxygenated by a CYP450 enzyme (**heme oxygenase**) to form **biliverdin** * Then it is reduced by **biliverdin reductase** to form **bilirubin** (both steps using NADPH... two in step 1... one in step 2)

Describe the _2nd phase_ biotransformation of heme. (include the _specific_ conjugating enzyme)

* **Bilirubin** is then _glucuronidated_ by **UDP-glucuronyl transferase** to form **bilirubin diglucuronide** (the transferase is specifically **UGT1A1**)

What transporter can bring _bilirubin_ or _bilirubin monoglucuronide_ into a hepatocyte? And which transports _bilirubin diglucuronide_ out of the cell?

entry - **OATP1B1** exit - **MRP2** (was pretty prominantly marked on a slide but not mentioned in lecture)

What are the 2 diseases which are _defects of conjugation_ in heme metabolism? Their causes + effects?

1. **_Gilbert syndrome_** - caused by mutation of promoter allele * slight elevation of unconj. bilirubin * mild jaundice * incr. paracetamol toxicity risk 2. **_Crigler-Najjar syndrome_** **-** UGT1A1 mutation * elevated (mostly) unconjugated bilirubin * intense jaundice * kernicterus (bilirubin-related encephalopathy) * incr. paracetamol toxicity risk

Which to diseases are defects of _conjugate export_ in heme metabolism? Their causes + effects?

1. **Dubin-johnson syndrome** - MRP2 defect * elevated conjugated bilirubin (w/out liver enzyme elevation) * dark pigment in liver * normal lifespan 2. **Rotor Syndrome** - defect unknown * elevated conjugated bilirubin * no liver pigmentation

What receptor is responsible for **CYP3A induction**? What binds it? What happens when it is bound?

**Pregnane X Receptor** - endogenous ligands = steroid hormones - forms _heterodimer_ with **retinoid X receptor** leading to upregulation of CYP3A

Describe the general structure of some of the nuclear receptors involved in CYP transcription regulation. Other than PXR mentioned in the last card, name 2 and their regulated CYP enzymes.

* are _ligand-dependent_ * contain a _double zinc finger_ **DNA binding domain (DBD)** * also have a larger **ligand binding domain** **(LBD**) CAR regulates CYP2B (constitutive androstane receptor) VDR regulates CYP27 (vitamin D receptor)

What molecule is important in the control of phase II enzymes? ## Footnote (didn't go over this slide in lecture... no clue abt it... just throwing it in)

**Nrf2**