Non microsomal enZymes

Phase 1 reaction. (Non synthetic phase). • Oxidation, reduction or hydrolysis. • OxidationMICROSOMAL ENZYMES- located in the Smooth endoplasmic reticulum in Liver and kidney, intestinal mucosa, lung • Eg:Monooxygenases, Cyp450. Glucoronyl transferases • Inducible by drugs, diet and other factors • Lesser polymorphism Phase II reaction. (Synthetic phase) NON MICROSOMAL Enzymes- in cytoplasm and mitochondria of hepatic cells and other tissues ( plasma) Eg: flavoprotein oxidases, esterases, amidases, conjugases, all conjugations ( except glucoronidation)(some ,reduction enzymes) Not inducible Genetic polymorphism (acetyltransferase,pseidocholi nesterase)

• •

NON MICROSOMAL : ENZYMES IN CYTOSOL (the soluble fraction of the cytoplasm):

• (a) Phase I: alcohol dehydrogenase, aldehyde reductase, aldehyde dehydrogenase, epoxide hydrolase, esterase.
• (b) Phase II: sulfotransferase, glutathione Stransferase, N-acetyl transferase, catechol 0methyl transferase, amino acid conjugating enzymes.

MITOCHONDRIA. • (a) Phase I: monoamine oxidase, aldehyde dehydrogenase, cytochrome P450. • (b) Phase II: N-acetyl transferase, amino acid conjugating enzymes.

LYSOSOMES. Phase I: peptidase.
NUCLEUS. Phase II: uridine diphosphate


• • • •

Esters, Amides, Hydrazides, and carbamates are hydrolyzed Hydrolysis in plasma mainly by cholinesterase (nonspecific acetylcholine esterases, pseudocholine esterases, and other esterases) In the liver by specific esterases for particular groups of compounds. Rate of Enzymatic hydrolysis of esters and amide: Role in the onset of pharmacological activity and its duration SUBCELLULAR LOCALIZATION. Endoplasmic reticulum and cytosol. TISSUE DISTRIBUTION. Ubiquitous, liver (centrilobular region), kidney (proximal tubules), testis, intestine, lung, plasma, and red blood cells.

SUBSTRATES. Esters and amides. REACTIONTYPE. Hydrolysis: • (a) Hydrolysis of esters: R1–CO–OR2 R1–COOH + R2–OH • (b) Hydrolysis of amides: R1–CO–NH-R2 R1–COOH + R2–NH2 • Hydrolysis of amides can occur by amidases in the liver and in general, enzymatic hydrolysis of amides is slower than that of esters. • Amides , also hydrolyzed by esterases with a much slower rate than the corresponding esters.

Carboxylesterases and cholinesterases
• Serine esterases • The catalytic site contains a nucleophilic serine residue- participates in hydrolysis of various xenobiotic, endobiotic substrates. • Carboxylesterases : serum, liver, intestine, and other tissues • Cholinesterases in blood (and muscles depending on the route of xenobiotic exposure) • Collectively determine the duration and site of action of certain drugs. • Eg: Procaine( ester—is rapidly hydrolyzed, a local anesthetic.) • Procainamide, the amide analog of procaine, ( hydrolyzed slowly; cardiac arrhythmia.) • The hydrolysis of xenobiotics by carboxylesterases , other hydrolytic enzymes is not always a detoxication process


• 60 kDa glycoproteins • Wide variety of tissues, including serum. ( in liver is associated with the endoplasmic reticulum, also in lysosomes and cytosol. ) • The hydrolysis of xenobiotic esters and amides : largely catalyzed by just two carboxylesterases called hCE1 and hCE2. • Human plasma does not contain carboxylesterases • Butyrylcholinesterases and Paraoxonases : responsible for the hydrolysis of amide and ester-containing compounds in the plasma • Also hydrolyze Endogenous compounds : palmitoyl-CoA, monoacylglycerol, diacylglycerol, retinyl ester, platelet- activating factor, and the synthesis of fatty acid ethyl ester ther esterified lipids. Mechanism : analogous serine-proteases. • A charge relay among : a.a. residue : glutamate ,histidine, nucleophilic residue :serine

Aldridge: Basis of their interaction with OP compounds, • A-esterases : hydrolyze OP compounds • B-esterases: inhibited by OP compounds • C-esterases : do not interact with OP compounds as. A Confusing : • because it divides the paraoxonases into the A- and C esterase class • The human paraoxonase hPNO1 hydrolyzes OP compounds and so can be classified as an A-esterase • hPON2 and hPON3 can be classified as C-esterases because they do not • hydrolyze OP compounds, nor are they inhibited by them) • Furthermore, carboxylesterases and cholinesterases, two distinct classes • of hydrolytic enzymes, are both B-esterases according to Aldridge because both are inhibited by OP compounds


• •

• • •

CHOLINESTERASES (AChE and BChE) Acetylcholinesterase (AChE) : High activity toward acetylcholine Butyrylcholinesterase (BChE, /Pseudocholinesterase): High activity toward acetylcholine and butyrylcholine (and propionylcholine) BChE can also hydrolyze: bambuterol, chlorpropaine, cocaine, methylprednisolone acetate, heroin, isosorbide diaspirinate, mivacurium, procaine, succinylcholine, tetracaine Eserine is an inhibitor of both enzymes BW84C51 is a selective inhibitor of AChE Iso-OMPA is a selective inhibitor of BChE

• Exist in six different forms with differing solubility in in three

states: soluble (hydrophilic), immobilized (asymmetric), and amphiphilic globular (membrane-bound through attachment to the phospholipid bilayer) • Six forms : monomer (G1), dimer (G2), tetramer (G4), tailed tetramers (A4), double tetramers (A8), and triple tetramers (A12).

• All forms are expressed in muscle. • AChE, major form in brain: tetramer G4 (anchored with a 20kDa side chain containing fatty acids)

• The major form in erythrocytes : the dimer G2 (anchored with a glycolipid-phosphatidylinositol side chain).
• BChE: major form in serum is the tetramer G4 (a glycoprotein with Mr 342 kDa). • In both AChE and BChE, the esteratic site (containing the active site serine residue) is adjacent to an anionic (negatively charged) site that interacts with the positively charged nitrogen on acetylcholine and butyrylcholine.

Carboxylesterases and cholinesterases • In blood and tissues play an important role in limiting the amount of OP compounds that reaches AChE in the brain • ChE inhibition : is the mechanism of toxicity of OP and carbamate insecticide • 70-90% loss of AChE activity is lethal to mammals, insects, and nematodes

• An inverse relationship between serine esterase activity and susceptibility to the toxic effect of OP compounds • Factors that decrease serine esterase activity potentiate the toxic effects of OP compounds • Eg-susceptibility of animals to the toxicity of parathion, malathion, and diisopropylfluorophosphate (DFP) is inversely related to the level of serum esterase activity (which reflects both carboxylesterase and BChE activity).

• Esterases are not the only enzymes involved in the detoxication of OP pesticides. • Certain OP compounds are detoxified by cytochrome P450, flavin monooxygenases,and glutathione transferases.

• Paraoxonases, enzymes that catalyze the hydrolysis of certain OP compounds, appear to play only a minor role in determining susceptibility to OP

POLYMORPHISM. • Approximately 2% of Caucasians have defective serum cholinesterase activity SPECIES DIFFERENCES • Activity is higher in small laboratory animals such as the rat and mouse than in humans.

PARAOXONASES (LACTONASES) • .Calcium-dependent enzymes containing a critical sulfhydryl (-SH) group; as such they are inhibited by EDTA, metal ions (Cu andBa), and various mercurials such as phenylmercuric acetate (PMA) • Catalyze the hydrolysis of a broad range of organophosphates, organophosphinites, aromatic carboxylic acid esters, cyclic carbonates, and lactones

Three paraoxonases : hPON1, hPON2, hPON3. hPON1 : • Liver microsomes and plasma, where it is associated exclusively with high-density lipoprotein (HDL) • protects against atherosclerosis by hydrolyzing specific derivatives of oxidized cholesterol and/or phospholipids in atherosclerotic lesions • Appreciable arylesterase activity and the ability to hydrolyze the toxic oxon metabolites of OPC insecticides • hPON2 : several tissues, not in plasma

• hPON3: serum and liver and kidney microsomes

• Luminal surface of the enterocytes lining the wall of the small intestine. • Hydrolysis of the prodrugs releasing the active drug at the surface of the enterocytes, where it can be readily absorbed. • Clinical applications in the treatment of certain cancers. • Eg: to activate prodrugs in vivo and thereby generate potent anticancer agents in highly selected target sites (e.g., at the surface of tumor cells, or inside the tumor cells themselves) • Eg: prodrugs, such as fosphenytoin and fosamprenavir

• The hydrolysis of valacyclovir to the antiviral drug acyclovir is catalyzed by a human enzyme named valacyclovirase (genesymbol: BPHL)

• Recombinant peptide hormones, growth factors, cytokines, soluble receptors, and humanized monoclonal antibodies : administered parenterally are hydrolyzed in the blood, Lysosomes and tissues by a variety of peptidases: Aminopeptidases and Carboxypeptidases • Hydrolyze amino acids at the N- and C-terminus, respectively • Endopeptidases, which cleave peptides at specific internal sites (trypsin, for example, cleaves peptides on the C-terminal side of arginine or lysine residues) • Peptidases cleave the amide linkage between adjacent amino acids, function as amidases. • ,The active site of peptidases : serine or cysteine residue, which initiates a nucleophilic attack on the carbonyl moiety of the amide bond.( like carboxylesterases)

NAD(P)H-dependent reductases
• Aldo–keto reductases (AKRs): Cytosolic enzymes that reduce both xenobiotic and endobiotic compounds, Function as dihydrodiol dehydrogenases and oxidize the trans-dihydrodiols of various polycyclic aromatic hydrocarbon oxiranes (formed by epoxide hydrolase)

Medium chain dehydrogenases/reductases (MDRs) : convert alcohols to aldehydes Eg: alcohol dehydrogenases

Short chainDehydrogenases/reductases (SDRs):

• Erythrocytic.cytosolic and microsomal carbonyl reductase, • reduction of a wide variety of carbonylcontaining xenobiotics (other species express more than two carbonyl reductases).

• Cytosol • reduction

• • Thioredoxin-dependent enzymes in liver and kidney cytosol Sulfoxide and N-Oxide Reduction: Reduce sulfoxides, which themselves may be formed by cytochrome P450 or flavin monooxygenases Eg: Sulindac is a sulfoxide that undergoes reduction to a sulfide, which is excreted in bile and reabsorbed from the intestine. • Reduction may also occur nonenzymatically at an appreciable rate, as in the case of the proton pump inhibitor rabeprazole

• 1953-Japan: The mechanism of lethal interaction b/w Sorivudine & 5-fluorouracil- involved inhibition of DPD – 15 deaths • An NADPH-requiring, homodimeric protein (Mr ∼210 kDa) containing FMN/FAD an liver cytosold an iron–sulfur cluster in each subunit. Location:, where it catalyzes the reduction of 5-fluorouracil and related pyrimidines. • Sorivudine is converted in part by gut flora to (E)-5-(2-bromovinyl) uracil (BVU), which lacks antiviral activity but which is converted by DPD to a metabolite that binds covalently to the enzyme. Resulting in irreversible inactivation(suicidal inactivation) of DPD • Marked inhibition of 5-fluorouracil metabolism, which increases blood levels of 5- fluorouracil to toxic lethal levels (\ • Genetic polymorphisms that result in a partial or complete loss of DPD activity

SUBCELLULAR LOCATION: Cytosol • Major enzyme responsible for oxidation of alcohol (ethanol) to aldehyde (acetaldehyde). Others: for ethanol oxidation:CYP2El ,Catalase, REACTION TYPE. • Oxidation of alcohol to aldehyde: R– CH2–OH R– CHO

SUBSTRATES. • Aliphatic or aromatic alcohols. COFACTORS. • NAD+. ENZYME STRUCTURE. • Zinc-containing dimer of two 40 kDa subunits. TISSUE DISTRIBUTION. • Liver, kidney, lung and gastric mucosa. POLYMORPHISM. 85% of Asians- class I isozymes (atypical ADH responsible for rapid conversion of ethanol to acetaldehyde), 20% of Caucasians - atypical ADH


ENZYME STRUCTURE. • Tetramer of 54 kDa subunits (ALDH1 and ALDH2) or dimer of 85 kDa subunits (ALDH) TISSUE DISTRIBUTION • . Liver, kidney, lung, and gastric mucosa SUBCELLULAR LOCATION. • Cytosol (ALDH1 and ALDH3), mitochondria (ALDH2) • Oxidation of xenobiotic aldehydes to acids. • In particular, acetaldehyde formed from ethanol by alcohol dehydrogenase is oxidized to acetic acid by ALDH, which is further oxidized to carbon dioxide and water. REACTION TYPE. • Oxidation of aldehyde to acid: R–CH2–CHO R–CH2–COOH

SUBSTRATES. • Aliphatic or aromatic aldehydes. COFACTORS : NAD+ or NADP+. POLYMORPHISM. • 50% of Asians have a defective ALDH2 gene causing impaired ALDH2 activitY


• Flavoprotein enzymes consists:two identical150 kDa subunits, each of which contains FAD, molybdenum, in the form of a pterin molybdenum cofactor ([MoVI(=S) (=O)]2+) and two iron–sulfur (Fe2S2) centers (known as FeSIand FeSII). An interaction between the molybdenum center with a reducing substrate Results in the reduction of the molybdenum cofactor, after which reducing equivalents are transferred intramolecularly to the flavin and iron–sulfur centers, with reoxidation occurring via the flavin moiety by molecular oxygen ALDEHYDE OXIDASE XANTHINE OXIDO REDUCTASE ( Xanthine dehydrogenase and Xanthine Oxidase) SULFITE REDUCTASE ( Oxidze sulfite, air poullutant)

• •

• • •

• Xanthine dehydrogenase (XD) and Xanthine oxidase (XO); Cytosol- Highest levels in heart, brain, liver, skeletal muscle, pancreas, small intestine, colon, and placenta • Two forms of the same enzyme that differ in the electron acceptor. XD: the final electron acceptor is NAD+ (dehydrogenase activity), XO : the final electron acceptor is oxygen (oxidase activity). • XD is converted to XO by oxidation of cysteine residues (Cys993 and Cys1326 of the human enzyme) and/or proteolytic cleavage. • Under normal physiologic conditions, XD is the predominant form of the enzyme found in vivo. • However, during tissue processing, the dehydrogenase form tends to be converted to oxidase form

XOR • Ischemia/Hypoxia: XO levels increase- XOR gene transcription, XD XO. • XO contributes to oxidative stress and lipid peroxidation because the oxidase activity of XO involves the reduction of molecular oxygen, which can lead to the formation of reactive oxygen species • LPS, a bacterial endotoxin that triggers an acute inflammatory response, increases XO activity both by inducing XOR transcription and by converting XD to XO.

XOR • First-pass elimination of purine derivatives (e.g., 6mercaptopurine and 2,6-dithiopurine), limits the therapeutic effects of • Certain prodrugs are activated by xanthine oxidase. Eg: antiviral prodrugs 6-deoxyacyclovir and 2_-fluoroarabinodideoxypurine, which are relatively well absorbed after oral dosing, are oxidized by xanthine oxidase to their respective active forms, acyclovir and 2--fluoroarabino-dideoxyinosine, which are otherwise poorly absorbed • Bioactivation of mitomycin C and related antineoplastic drugs, although this bioactivation • Catalyzes, the sequential oxidation of hypoxanthine to xanthine and uric acid

• Allopurinol: Hydroxylated coumarin derivatives, such as umbelliferone (7hydroxycoumarin) and esculetin (7,8dihydroxycoumarin • By competing with hypoxanthine and xanthine for oxidation by XD/XO, inhibits the formation of uric acid,. • Monomethylated xanthines ( except theophylline and caffeine) oxidized to the corresponding uric acid derivatives by XD/XO.

• Exists only in the oxidase form as it lacks an NAD+ binding site • High levels: in cytosol of liver, with considerably less activity in other tissues • Preference for oxidizing aromatic aldehydes ( benzaldehyde) over aliphatic aldehydes. (acetaldehyde) • Transfers electrons to molecular oxygen, which can generate reactive oxygen species and lead to oxidative stress and lipid peroxidation. • Oxidize a number of substituted pyrroles, pyridines, pyrimidines, purines, pteridines, and iminium ions • plays an important role in the catabolism of biogenic amines and catecholamines: physiologically important aldehydes- substrates: homovanillyl aldehyde (formed from dopamine), 5-hydroxy-3indoleacetaldehyde (formed from serotonin), and retinal, which is converted to retinoic acid • In general, xenobiotics that are good substrates for aldehyd e oxidase are poor substrates for cytochrome P450, and vice versa

• Species difference : Dogs possess little or no aldehyde oxidase activity. • 6-oxidation of antiviral deoxyguanine prodrugs is catalyzed exclusively in rats by XD/XO, but by aldehyde oxidase in humans • Drugs metabolized: Nicotine, citalopram, proprionaldehyde, 6-mercaptopurine, metyrapone, quinine, methotrexate, famciclovir ( to penciclovir) • Raloxifene and perphenazine : potent inhibitors • Under certain conditions, aldehyde oxidase and XOR can also catalyze the reduction of xenobiotics, including azo-reduction (e.g., 4-dimethylaminoazobenzene), nitro-reduction (e.g., 1nitropyrene), N-oxide reduction (e.g., S-(-)-nicotine-1-N-oxide), nitrosamine eduction (e.g., N-nitrosodiphenylamine),sulfite reduction

MAO, DIAMINE OXIDASE and POLYAMINE OXIDASE SUBSTRATES. • Primary, secondary, and tertiary naturally occurring .amines: • Monoamines: serotonin (5-hydroxytryptamine) • Diamine: putrescine and Monoacetylated derivatives of the polyamines spermine and spermidine. • Oxidative deamination(Primary amine)—ammonia & aldehyde • Oxidative deamination (Secondary) -- primary amine and an aldehyde. (The products of the former reaction—i.e., an aldehyde and ammonia—are those produced during the reductive biotransformation of certain oximes by aldehyde oxidase) • The aldehydes formed are usually oxidized further by other enzymes to the corresponding carboxylic acids, although in some cases they are reduced to alcohols.

• Related to the metabolism of exogenous tyramine and the “cheese effect” produced as a result of the ingestion of large amounts of tyramine-containing foods • Catalyzes the oxidative deamination of biogenic amines TISSUE DISTRIBUTION. • Ubiquitous,throughout the brain, and is in outer membrane of mitochondria the liver, kidney, intestine, and blood platelets lymphocytes. SUBCELLULAR LOCATION. : Mitochondria, some MAO activity in the microsomal fraction. REACTION TYPE. • Oxidative deamination of amines: RCH2–NR1R2 R–CHO + NHR1R2

MAO Substrates ; drugs- milacemide (dealkylated metabolite of propranolol ) , , primaquine, haloperidol, doxylamine, β-phenylethylamine, tryptophan analogs known as triptans: sumatriptan, zolmitriptan, and rizatriptan. Endogenous: tyramine, catecholamines (dopamine, norepinephrine, epinephrine), tryptophan derivatives (tryptamine, serotonin), Isoforms : MAO-A and MAO-B. OXIDIZE INHIBITED BY MAO-A serotonin (5-hydroxytryptamine) clorgyline norepinephrine phenelzine , metabolite of propranolol, MAO-B β-phenylethylamine l-deprenyl (selegiline). benzylamine phenelzine • Species differences in the substrate specificity of MAO -dopamine is oxidized by MAO-B in humans, but by MAO-A in rats, and by both enzymes in several other mammalian species. • Most tissues contain both forms of the enzyme, each encoded by a distinct gene, although some tissues express only one MAO.


• The activation of MPTP (1-methyl-4-phenyl1,2,5,6-tetrahydropyridine)to its neurotoxic metabolite is catalyzed predominantly by MAO – B: • haloperidol- to toxic pyridinium • Parkinson’s disease in humans: elevated levels of MAO-B( dopamine destruction)

• Cytosolic, copper-containing, pyridoxal phosphatedependent enzyme present in liver, kidney, intestine, and placenta. • Substrates: Histamine and simple alkyl diamines with a chain length of 4 (putrescine) or 5 (cadaverine) carbon atoms. • Diamines with carbon chains longer than 9 are not substrates for DAO, although they can be oxidized by MAO. • DAO/ similar enzyme is present in cardiovascular tissue cardiotoxic effects of allylamine, which is converted by oxidative deamination to acrolein. • No DAO in brain : The major pathway of histamine metabolism in the brain is by methylation

SEMICARBAZIDE-SENSITIVE AMINE OXIDASE (SSAO) • Copper-containing enzyme that catalyzes fundamentally the same reaction catalyzed by monoamine oxidase: • Distinguished from MAO: By its sensitivity to inhibitors (it is inhibited by semicarbazide but not by clorgyline, deprenyl, or pargyline, whereas the opposite is true for MAO), and • it is found on various cell surfaces and in plasma, (MAO- found in mitochondria).

• TISSUE DISTRIBUTION. Liver, kidney, adrenals, lung, brain, jejunum, and blood platelets, and, to a lesser extent, skin and muscle. • SUBCELLULAR LOCATION. Cytosol. •

Homodimers in vivo with a molecular weight 32-34 kDa. • Mediates conjugation reaction of a compound at a low concentration • In General a high-affinity and low-capacity reaction . REACTION TYPE. Sulfation: (a) O-sulfation: R-OH R-SO3H (b) N-sulfation: R-NHCOR R-NCOR SUBSTRATES. SO3H • Nucleophilic moieties of such molecules as phenol, alcohol, and arylamine. COFACTOR. 3´-Phosphoadenosine-5´-phosphosulfate (PAPS).

ISOZYMES. • Six different phenol sulfotransferases (PST • Seven different steroid/ bile acid sulfotransferases have been characterized in rats. In human • Four subfamilies: TS ST (thermostable ST or PST), TL ST (thermolabile ST, or monoamine ST), EST (estrogen ST),DHEA ST (dehydroepiandrosterone ST). POLYMORPHISM. • Bimodal frequency distribution of DHEA ST activity suggests that approximately 75 % of the population are poor metabolizers SPECIES DIFFERENCES. • The pig and opossum are defective in their capability regarding sulfate conjugation of phenolic compounds.

RELATIONSHIPS BETWEEN UDPGT AND ST • Often, UDPGT and ST are considered to be complementary to each other for conjugation of the same substrates, except in connection with acyl glucuronidation, which cannot be replaced by sulfation. • In general, glucuronidation is considered a low-affinity (Km) and high-capacity (Vmax) reaction, whereas sulfation is known as a high-affinity and • low-capacity conjugation. • Thus, at low substrate concentrations sulfation may be more predominant, but as concentration increases, glucuronidation becomes quantitatively more important.

• The first genetic polymorphism described for an enzyme involved in human drug metabolism was for NAT • Molecular weight of 26.5 kDa TISSUE DISTRIBUTION. • Liver (in the Kupffer cells, not in the hepatocytes), spleen, lung, intestine. SUBCELLULAR LOCATION. : Cytosol. REACTION TYPE.: Acetylation on amine moiety. R- NH2 R-NH-COCH3 R-SO2-NH2 R-SO2NH-COCH3 SUBSTRATES. Aromatic amines (R–NH2),sulfonamides (R–SO2–NH2), or hydrazine (R–NH–NH2) derivatives. COFACTOR. : Acetyl-coenzyme A(CoA).

N-ACETYL TRANSFERASE (NAT) ISOZYMES. • NAT1 and NAT2 enzymes POLYMORPHISM. • 40–60% of Caucasians and 10–30% of Asians are slow acetylators. SPECIES DIFFERENCES. • Dogs and guinea pigs are deficient

• Integral part of the phase II detoxification system. • Protects cells from oxidative- and chemical-induced toxicity and stress by catalyzing the glutathione conjugation reaction with an electrophilic moiety of lipophilic and often toxic xenobiotics REACTION TYPE. Glutathione conjugation. R–X R–S–glutathione + X- (X: halide, sulfate, or phosphate) R–C = C–COR R–C–C–COR S–glutathione ENZYME STRUCTURE. • Dimer in vivo with a molecular weight of 24–28 kDa. TISSUE DISTRIBUTION. • Liver, gut, kidney, testis, adrenal, and lung. SUBCELLULAR LOCATION . Cytosol (major) and endoplasmic reticulum (minor).

ISOZYMES. • Five classes of cytosolic enzymes, α, μ, π, 0- , and σ and one class of microsomal enzyme. POLYMORPHISM: GSTM1 (the class μ enzyme): 40–50% of individuals; GSTT1 (the class θ enzyme): 10–30% of Europeans have a deficiency. SUBSTRATES. • Lipophilic and have an electrophilic moiety. • Glutathione conjugates of xenobiotics. In liver are usually excreted in bile and urine/ further metabolized to mercapturic acids(kidney, excr. Urine). • Substrates with reactive or good leaving groups : epoxide, halide, sulfate, phosphate, or nitro moiety attached to an allylic or a benzylic carbon. • Facilitated by electron-withdrawing groups, such as – CHO, –COOR, –COR, or –CN, adjacent to the electrophilic moiety of the compounds. COFACTOR: Glutathione, a tripeptide cofactor (GSH, L-y-glutamyl-Lcysteinylglycine (Gly-Cys-Glu)), is present in virtually all tissues, often in relatively high (0.1– 10mM) concentrations.

• Methylation of endogenous substrates such as histamine, catecholamines, and norepinephrine. And some drugs COFACTOR.: S-adenosylmethionine(SAM). TISSUE DISTRIBUTION: • Liver, brain, lung, kidney, adrenals, skin, and erythrocytes. SUBCELLULAR LOCATION.: Cytosol. ISOZYMES.: Four different enzymes can perform S-, N-, or 0methylation POLYMORPHISM. 0.3% of the European population have a deficiency in thiopurine S-methyltransferase activity. • In general, methylation of a compound produces a less polar metabolite than the parent compound, and thus, unlike other conjugation reactions, tends to decrease the rate of its excretion.

REACTION TYPE. 0, N, S-methylation:

0, N, S-methylation

• Exogenous carboxylic acid, ( acetates), activated to coenzyme A derivatives (acyl CoA thioether) in vivo by acyl-CoA synthetase
• Further conjugated with endogenous amines such as amino acids by acyl-CoA:amino acid N-acyltransferase; Amino acid conjugates eliminated primarily in urine by tubular active secretion mechanisms

COFACTORS. • Coenzyme A (CoA-SH) for acyl-CoA synthetase • Amino acids: glycine, glutamine, ornithine, arginine, and taurine, for acylCoA:amino acid N–acyltransferase. TISSUE DISTRIBUTION. Liver and kidney. SUBCELLULAR LOCATION. : Mitochondria and endoplasmic reticulum for acyl-CoA synthetase, and cytosol and mitochondria for acyl-CoA:amino acid N-acyltransferase. SPECIES DIFFERENCES: The amino acid used for conjugation • Eg: conjugation of bile acids occurs with both glycine and taurine in most species, whereas in cats and dogs, conjugation of bile acids occurs only with taurine