|Lecture Notes:: 9 March||
Reductions take place in both the soluble and microsomal fractions under anaerobic conditions. Cytochrome P450 or a flavoprotein may be involved in the microsomal reactions, with NADPH as a required reductant.
Important reduction reactions may also take place in the gut by bacterial P450 reductases. An example is the reduction of the azo dye prontosil to give the antimicrobial drug sulfanilamide:
Nitrobenzene toxicity to red blood cells occurs as a result of its reduction by the microbial P450/NADPH system. The nitrosobenzene and phenylhydroxylamine intermediates in this reduction are toxic to red cells:
Under anerobic conditions can get dehalogenation using P450/NADPH. An example is the reaction of Halothane under anaerobic conditions:
Esters and amides are commonly hydrolyzed:
An example is the hydrolysis of the anesthetic, procaine, that is rapidly hydrolized by esterases in the plasma and by tissue microsomal esterases:
The analogous amide, procainamide, is hydrolyzed more slowly by amidases in the microsomal fraction of tissue. It is not hydrolyzed at all in the plasma.
Phase Two Reactions
Phase two reactions involve the attachment of a generally polar, readily available in vivo, molecule to a susceptible functional group. Reactive functional groups are often, but not necessarily a result of Phase 1 reactions). The result is to make the whole molecule more polar, and thus more readily excreted.
Process in vivo reactant Product Glucuronide formation
Glucuronide (X = O, N, or S) Glutathione conjugation
glutathione (Glu, Cys, Gly)
Mercapturic acid Sulfate conjugation
PAPS = Adenosine-3'-P-5'-Phosphosulfate
Aryl or alkyl sulfates and sulfamates Acetylation
Acetyl Coenzyme A
Acetylated derivatives Amino acid conjugation Coenzyme A, ATP & e.g. glycine Acyl derivatives Methylation
Let's look at examples for these different Phase 2 processes.
Glucuronide Formation: In order to form glucuronides we must first synthesize UDP-glucuronic acid by activating and oxidizing glucose.
- Glucose UDPG
Glucose + ATP Glucose-6-P + ADP
G-1-P + UTP UDPG + PPi where PPi = inorganic pyrophosphate
PPi 2 Pi where Pi = inorganic phosphate
Note that the hydrolysis of PPi drives the reaction, since it is very favorable and the product of the previous reaction is removed. These rections take place in the cytosol.
- Conjugation by glucuronyl transferase now takes place on the ER, mostly via reaction with hydroxyl and carbonyl groups (but occasionally with N & S also). Generally we see SN2 reactions, so the -UDPG yields -glucuronides.
Example: Conjugation of phenol gives an acetal glucuronide, phenyl--D-glucuronide:
Conjugation with benzoic acid gives an ester glucuronide, benzoyl--D-glucuronide:
Examples with nitrogen and sulfur include the conjugation of aniline to give phenylamino--D-glucuronide, and conjugation with 2-mercaptobenzothiazole to give the benzothiazole-2-thio--D-glucuronide:
Glutathione conjugation: Glutathione is a tripeptide (-glutamylcyteinylglycine, with the amino acid residues color coded in the structures below). It is used in cells as a recyclable anti-oxidant as well as a conjugating agent. When used in conjugation it is initially conjugated by a glutathione-S-transferase, or sometimes as a simple chemical reaction. Generally the conjugation occurs as a nucleophilic attack by the -SH group on a reactive electrophilic center. Conjugation is often followed by metabolic cleavage of the peptide to leave only the cysteinyl residue, which may then be acetylated to give a mercapturic acid.
1. Glutathione S-transferase:
3. cysteinyl glycinase:
4. N-acetyl transferase:
Of course not all glutathione derivatives are further metabolized, some may be excreted as is. However, if the derivative is excreted via the bile, it may be metabolized by the gut fauna and the toxin may be reabsorbed.
Sulfate Conjugation: This is the major Phase two process for a variety of hydroxyl groups and for some amines.
- The first step is to "activate" the sulfate to give "PAPS" (Adenosine-3'-P-5'-Phosphosulfate)
These enzymes are in the soluble fractions of a wide variety of tissues (e.g. liver, muscle, intestine, testes, etc.).
- Can get Aryl sulfates, Alkyl sulfates, and Sulfamates via a sulfotransferase reaction.
Example, sulfamate synthesis
Acetylation: Acetyl transferases are found in the cytosol of hepatic endothelial cells, intestinal mucosal cells and white blood cells.
The "activated" acetyl group on acetyl CoA is used as the source of acetate, so it is readily available from normal metabolic sources. The enzyme mechanism is outlined below:
Example: acetylation of a primary aryl amine:
There is evidence for more than one acetylase, which provides the genetic basis for "slow" and "fast" acetylators. Apparently there is polymorphism for various acetylases (we will see again later in comparative studies).
Slow acetylators are more susceptable to xenobiotics which are inactivated by acetylation. Turns out deacetylases are also present. The two processes show dominance in different organisms, varying the importance of acetylation.
Acylation: Acylation is similar to acetylation in the products excreted, but with an amino acid substituting for acetate (most commonly glycine; ornithine, taurine, and glutamine are also known). However, the synthetic strategy is "reversed" in that the xenobiotic is activated by adding coenzyme A, which then reacts with the amino acid.These reactions may be catalyzed by fatty acyl CoA synthetases:
This enzyme catalyses a two part reaction:
The resulting acylCoA derivative can then react with an amino acid to give the acyl excretory product.
Example: Benzoic acid is first converted to benzoyl CoA, the benzoyl CoA then reacts with glycine to give the product, Hippuric acid:
Last modified 9 March 2010
© RA Paselk 2001