PHASE II--Conjugation Reactions

A. Glucuronidation--Major pathway in most animals except cats

1. Requires cofactors UDGA

2. located in ER (facing lumen)

3. substrates
--chemical that contains electron rich nucleophilic (heteroatom) O, N or S;
some extremely electophillic carbons

--endogenous substrates--bilirubin, steroids, thyroid hormones (in rat)

4. Site of elimination of conjugate (size)

5. Why Glucuronic Acid??

6. Synthesis of cofactor

7. Structure

--C-terminus spans membrane of ER (anchors)

--faces lumen; low detergent concentrations may increase activity

8. Multiple isoforms--2 Gene families UGT1 and UGT2

a. UGT1--members are formed by alternate splicing of a single gene

1. links different substrate binding sites to a constant portion of

enzyme

2. UGT1.6--3MC inducible conjugates planar molecules

3. UGT1.1--PB inducible

4. UGT1.4 clofibric acid-inducible

5. PCN-inducible---->dt1
 b. UGT2 <50% aa identity with UGT1

1. 2 subfamilies UGT2A (olfactory epithelium) and UGT2B (liver)

2. UGT2B1, 2,3,6,12 liver of rat

a. 2, 12 are inducible by PB

b. UGT2B4, 7, 8, 9, 10, 11--human

c. UGT2B5-mouse

d. UGT2B13,14 --rabbit

9. Distribution in Human liver

--UGT1--6 isoforms

--UGT2B4, 7, 8, 9, 10, 11

10. Genetic Defects

 a. Grigler-Najjar syndrome

1. Type I mutation in exons 2-5 loss of 1.4 and 1.1 and bilirubin
conj.

2. Type II mutation in exon 1--decreased activity of 1.1 (not absent)

b. Gilberts disease (5-7%) --hyperbilirubinema

11. Relationship to Toxicity

a. UGT1.6 coinduced with CYP1A2

b Gilberts disease prone to hepatotoxicity by acetaminophen

c. Thyroid tumor in rats

d. aromatic amines

e. acyl glucuronides
B. Sulfation

1. Location-cytosolic; liver, kidney, intestinal tract, lung platelets, brain

2. Cofacter-3'phosphoadenosine-5'-phosphosulfate (PAPS)

3. substrates

a. carboxylic acids cannot be conjugated with SO3-, but can be competitive

inhibitor

b. pentachlorophenol; 2,6-dichloro-4-nitrophenol (DCNP)--->ST inhibitors

(bind enzyme, but not nucleophilic enough to attack PAPS)

4. conjugates mainly excreted in urine

a. if biliary--->aryl sulfatases (bacteria)

b. sulfatases also present in ER and lysosomes

5. PAPS synthesis
ATP sulforylase
a. cys--->inorganic sulfate + ATP-----------------------> APS
APS kinase
b. APS + ATP --------------------> PAPS

6. 5 Classes (>12 isoforms in rat)

a. ArylST

b. Alcohol ST

c. Estrogen ST

d. Tyrosine ester ST

e. Bile salt ST
7. 3-purified isoforms from human liver cytosol

a. 2---phenol transferases (PSTs)

1. thermally stable TS-PST---simple phenols; inhibited by DCNP

a. expression--genetic

2. thermally labile TL-PST--monoamines (dopamine, EPI, L-

DOPA) not inhibited by DCNP;

b. third enzyme Alcohol ST------->DHEA (dehydroepiandrosterone)

resistant to DCNP

1. bimodal distribution 25% of caucasians high activity

c. 4th enzyme cloned estrogen ST

8. Toxicity associated with sulfation

C. Methylation

1. Cofacter--SAM S-adenosyl methionine

a. methyl group has characteristics of carbonium ion

2. Substrates

a. also metals Hg; As; Se

3. O-methylation of phenols----POMT microsomal

4. O-methylation of catechols----COMT cytosolic

a. present in all tissues (liver and kidney)

b. neurotransmitters, catechols, estrogens

c. encoded by single gene in humans

1. Low activity form (COMTL) and high activity form (COMTH)

a. 25% of caucasians are homozygous for low or high

b. 50% are heterozygous--->intermediate

2. 75% of African americans L-DOPA

 5. N-methyltransferase (2 isoforms in humans)

a. histamine N-methyltransferase (imidazole ring)

1. in RBCs under genetic control (varies 6-fold)

b. nicotinamide N-methyltransferase (pyridine rings--nicotine; indole rings--

tryptophan)

6. S-methylation

a. 2 enzymes in human

1. Thiopurine methyltransferase (TPMT)

a. cytosolic--aromatic/heterocyclic

b. encoded by single gene

2. Thiol methyltransferase (TMT)

a. microsomal--aliphatic sulfhydryls

b. H2S from bacteria---->methyl-SH------>DMS

c. methylates glutathione conjugates following β-lyase

D. Acetylation

1. Substrates--aromatic amines; hydrazines

2. Cofacter-Acetyl-Coenzyme-A

3. location--cytosolic (liver and other tissues)--except dogs and foxes

4. 2 isoforms in Human

a. NAT1 and NAT2---79-95% conserved in all species cys68 in active site

b. independently regulated NAT1 in most tissues; NAT2 liver and gut

only
c. different but overlapping substrate specificites

d. Genetic polymorphism

1. Middle East (Egyptian/Saudi Arab./Moraccans)

2. Caucasians (US/Austrailia/European)

3. Asian (Japanese/Chinese/Korean)

e. Toxicities associated with NAT

1. detox of AA---->amides

2. activation of AA---> N--->O transfer (O-acetyltransferase)

a. poor acetylators susceptible to bladder cancer (bicylic

AA)

b.fast acetylators susceptible to colon cancer, but protected

from bladder cancer

1. O-acetylation of N-OH heterocyclics (NAT2)

whereas bicyclics are acetylated by NAT1 and NAT2
in liver (detox); heterocyclics are poor substrates for
N-acetylation by NAT1 and NAT2

2. CYP1A2--->activates AA--->N-OH--AA
---->activated by bladder NAT1 (O-acetylation)
 3. increases in NAT2 does little to prevent CYP1A2
activation of heterocyclics (poor substrates for
NAT2) in liver
a. Gut NAT2 then O-acetylates in colon

N-acetylation and O-acetylation can be reversed by aryl acetamide deacetylase (microsomal)

E. Amino acid Conjugation

1. Conjugation of carboxylic acids with amino groups of gly, glut, taurine

a. activation of xenobiotic with CoA conjugation

1. acyl-CoA thioester reacts with amino group of aa to form an

amide linkage

2. Conjugation of hydroxylamines with carboxylic acid group, ser, pro

a. activation of amino acid by aminoacyl-tRNA-synthetase (cytosolic)

1. reacts with aromatic hydroxylamine to form reactive N-ester

3. Conjugation of benzoic acid with glycine---->hippuric acid 1842

4. Bile acids are endogenous substrates for gly, taurine (occurs in microsomes)
 a. xenobiotics----->mitochondria (multiple acy CoA synthetases

b. Xeno--2nd step--cytosolic and/or mitochondria N-acyl-transferase

1. 2 forms of mammalian hepatic mitochondria enzyme

a. benzoyl-CoA as substrate

b. arylacetyl-CoA

c. Bile acids---->bile; aa-conj of xenos--->urine

5. Receptor acids (substrate and species specific)

a. benzoic, hetocyclic, cinnamic acis---->gly

b. Aryl acetic acids--->gly (primates--glut)

c. mammals---taurine alternative for gly

6. Alternative to glucuronidation for carboxylic acids----thus is always detox

7. aa conjugation of hydroylaromatic amines--->activation

F. Glutathione Conjugation--->gly, cys, glutamic acid (attached by γ carbonyl group)

1. General features

a. product is thioether (not amide)

b. thiolate anion (nucleophilic) attacks multitude of electrophilic substrates

c. conjugates electrophilic heteroatoms

2. Synthesis of GSH

a. γ-glutamylcystein synthetase

b. glutathione synthetase

3. Conjugation can occur spontaneously or through GSTs

a. GSTs present in most tissues 95% found in cytosol 5% in microsomes

4. Substrate features

a. hydrophobic

b. electrophilic

c. react nonenzymatically with GSH at some measureable rate

5. Enzymatic mechanism

a. deprotonation of GSH---->GS - by active site tyrosinate (general base

catalyst).

b. stereoselectivity (although some nonenzymatic--most enzymatic)

c. enzyme accounts for up to 10% of total cellular protein

d. Binds stores, transports (heme, bilirubin, steroids, azo-dyes, PAHs)

6. Enzymatic substrates

a. direct conjugation

b. B/T by Phase I/II electrophile that is then conjugated

1. oxiranes

2. nitrenium

3. carbonium

4. free radicals

7. Enzymatic reactions
a. Displacement reactions (displacement of e- withdrawing group)

1. substrate allylic leaving group

2. reduced if electron donating group present on aromatic ring

3. increased if electron withdrawing group present on aromatic

a. 1,2-dichloro-4-nitrobenzene (DCNB)

b. 1-chloro-2,4-dinitro benzene (CDNB)

4. O-demethylation of dimethylvinphos and other Me-OPs

b. addition reactions C-C double bond

1. increased by adjacent electron withdrawing group

2. DEM; NABQI

3. arene oxides---stereoselectivity
 4. Electrophilic heteroatom-

a. requires 2 molecules of GSH

b. reduction of hydroperoxides by GST refered to as non-

selenium-requiring GSH Px

8. Fate of conjugates

a. biliary GS-conjugates

b. Transported to kidney------>mercapturic acids---->

1. sequencial cleavage of glutamic acid and gly (γ-glutamyl

transpeptidase) (aminopeptidase M)
 2. N-acetylation

9. Structure of GSTs (cytosolic)

a. nomenclature based on subunits; class membership requires >

50% identity

1. Alpha

a. humans 2 subunits GSTA1 and GSTA2

b. rats 4 GSTA1 = ligandin

c. major forms in liver and kidney (basic isoelectric

points)

2. Mu

a. humans 4 subunits GSTM1a, M1b, M2 and M3

b. rats 5 subunits GSTM1-5

c. neutral isoelectric points

 3. Pi

a. humans--2 subunits; GSTP1a; P1b

b. rats--1 subunit GSTP1 (overexpressed in

chemical induced tumors)

c. acidic isoelectric points; placenta, lung, gut,

extrahepatic

4. theta

a. humans and rats --1 form; ancestral gene

10. Microsomal GST

a. trimeric enzyme--->xenos

b. LC 4 synthase

11. Assays for class identity

a. CDNB---α, µ. π

b. α
1. isomerization of δ5steroids to δ4 steroids

2. reduce linoleate and cumene hydroperoxide

c. µ---arene oxides; alkene epoxides

d. π--ethacrynic acid

12. Regulation

a. α and µ are 2-3 fold inducible by 3MC, PB, corticosteroids, and

antioxidants

b. induction---> increased mRNA (transcriptional activation)

1. XRE, barbie box, GRE, ARE

2. monofunctional and bifunctional regulation

c. Drug and pesticide resistance

 13. Role in Toxicity

a. Mice resistant to AB1 tumors;

b. Genetic polymorphism GSTM1

c. Increasing toxicity

1. formation of GSH conjugates of haloalkanes, organic

thiocyanates and nitroguanides that release a toxic metabolite

2. formation of GSh conjugates of vicinal dihaloalkanes that

form sulfur mustards

3. formation of GSH conjugates of halogenated alkenes

degraded to toxic metabolites by β-lyase in kidney

4. formation of GSH conjugates of quinones,

quinoneimines, and isothiocyanates that are degraded to
toxic metabolites by γ-GT in kidney

G. Rhodanese (mitochondrial)

1. cyanide to thiocyanate