Phase 3 Reactions

Question 1

What are Phase 2 reactions usually catalysed by?

Answer 1

Transferase enzymes

Transferring a polar group from a donor or conjugating agent to the phase 1 metabolite

Question 2

What enzyme catalyses glucuronidation?

Answer 2

Glucuronosyltransferase

UDP - glucuronyltransferase (UGT)

Question 3

What enzyme catalyses sulphation?

Answer 3

Sulphotransferase

Question 4

What enzyme catalyses amino acid conjugation?

Answer 4

N-acyl transferase

Question 5

What enzyme catalyses Acetylation?

Answer 5

N-Acetyltransferase

Question 6

What enzyme catalyses methylation?

Answer 6

Methyltransferase

Question 7

What enzyme catalyses glutathione conjugation?

Answer 7

Glutathione S-transferase

Question 8

What is glutathione conjugation important?

Answer 8

Important for detoxification

Question 9

Where is UGT found?

Answer 9

Liver, intestine, kidneys, brain, adrenal glands, spleen and thymus

Question 10

What is the donor molecule for glucuronidation? Why?

Answer 10

UDPGA - Uridine Diphosphate Glucuronic Acid

High energy
Abundant

Also related to glycogen synthesis - endogenous

Question 11

What organelle is UGT found in?

Answer 11

Smooth endoplasmic recticulum

Question 12

What does UGT do?

Answer 12

Transfers Glucuronyl group from UDPGA to nucleophilic O, N or S

Question 13

What’re the similarities and differences between UGT1 UGT2 and UGT3?

Answer 13

All 3 proteins contain similar region which recognises UDP glucuronic acid

Each proteins has a different region for substrate recognition

Question 14

Which 2 UGT enzymes are the key subtypes involved in drug glucuronidation?

Answer 14

UGT1 and UGT2

Question 15

Give an example of O-glucuronidation.

Answer 15

Salicylic acid

Both -COOH and -OH groups can undergo glucuronidation (can be doubly conjugated)

Question 16

Give an example of N-glucuronidation.

Answer 16

Sulphanilamide

UDPGA coordinates with both NH2 groups, this is recognised by UGT

Both sites are then glucuronidated

Question 17

What is the donor molecule for sulphation? Why?

Answer 17

PAPS - 3’-phosphoadenosine-5’-phosphosulfate

Energy rich

Question 18

What is the cytosolic transferase responsible for sulphation?

Answer 18

Sulphotransferase

Question 19

What predominates at low substrate concentration, sulphation or glucuronidation? Why?

Answer 19

Sulphation

Because there is relatively less PAPS in cell cytosol compared to UDPGA

Question 20

What predominates at high substrate concentration, sulphation or glucuronidation? Why?

Answer 20

Glucuronidation

Because there is relatively less PAPS in cell cytosol compared to UDPGA

Question 21

Give an example of sulphation that stops Phase 1 metabolism.

Answer 21

Paracetamol

-OH is sulphonated which stops Phase 1 metabolism

It is then excreted, build up causes toxicity issues

Question 22

Where does glycine conjugation occur?

Answer 22

In the mitochondria

Question 23

What is the donor molecule for glycine conjugation?

Answer 23

Glycine (glutamine, taurine)

No HIGH ENERGY donor molecule

Activated to form Acyl CoA intermediate

Question 24

How does a drug become activated prior to glycine conjugation?

Answer 24

-COOH reacts with ATP to form C(O)-AMP

-C(O)-AMP reacts with Coenzyme-A synthetase to form -C(O)-S-CoA

Then reacts with glycine via acyl Coenzyme-A thioester intermediate

Question 25

What enzyme catalyses glycine conjugation?

Answer 25

N-Acyl transferase

Question 26

Why is glutathione conjugation important?

Answer 26

As it attacks epoxides and quinoneimines These are reactive metabolites

Question 27

What enzyme catalyses glutathione conjugation?

Answer 27

Glutathione S-transferase - GST

Question 28

What is the high energy donor for glutathione conjugation and where is GST found?

Answer 28

Found in the cytosol of hepatocytes No high energy donor as substrates are already chemically reactive

Question 29

What can glutathione conjugated metabolites undergo further metabolism to?

Answer 29

Further metabolism to mercapturic acids

Question 30

Give an example of a quinoneimine being produced from Phase 1 metabolism

Answer 30

Paracetamol is oxidised by CYP450 enzyme to form N-acetyl-p-benzoquinoneimine (NAPQI) which is very reactive and hence bad It then undergoes glutathione conjugation to make it more hydrophilic and can then be excreted

Question 31

What is often referred to as Phase 3 metabolism of drugs?

Answer 31

Mercapturic acid synthesis It is an inter-organ pathway involving the liver and kidneys

Question 32

When does Phase 3 metabolism occur?

Answer 32

ONLY after glutathione conjugation reactions done in Phase 2

Question 33

What’s the predominant reaction for primary amines and sulphonamides?

Answer 33

Acetylation and sulphation

Question 34

What is the high energy donor for acetylation?

Answer 34

Acetyl Coenzyme-A

Question 35

What can happen as a result of acetylation?

Answer 35

Acetylation often decreases water solubility and hence it can precipitate out in the kidneys leading to renal toxicity

Question 36

What are commonly methylated functional groups?

Answer 36

Phenols Catechols Amines N=heterocycles sulfhydryls

Question 37

What does methylation require the presence of?

Answer 37

Cofactor - a methyl donor S-adenosyl methionine (SAM)

Question 38

What 2 specific enzymes catalyse methylation?

Answer 38

Catechol-O-methyltransferase - COMT - O-methylation Thiopurine S-methyltransferase - TPMT - S-methylation

Question 39

How is 6-mercaptopurine metabolised for excretion?

Answer 39

Metabolised to methyl 6-mercaptopurine in the presence of TPMT and SAM

Question 40

How does 6-mercaptopurine become pharmaceutically active?

Answer 40

It is oxidised twice by xanthine oxidase to 6-thiouric acid This is cytotoxic and kills cancer cells

Question 41

Give an example of a methylation reaction COMT catalyses.

Answer 41

L-DOPA, both -OHs can be methylated In the presence of SAM ofc

Question 42

What metabolism pathways commonly occur to -OH?

Answer 42

Glucuronidation Sulphation Methylation

Question 43

What metabolic pathways commonly occur to -COOH?

Answer 43

Glucuronidation Glycine conjugation

Question 44

What metabolic pathways commonly occur to -NH2?

Answer 44

Acetylation Glucuronidation Sulphation