Phase II Metabolism (Chris)

General Information
Phase 2 metabolic reactions are conjugation reactions catalyzed by transferase enzymes1. Unlike the phase 1 reactions, which usually introduce polar groups, the phase 2 reactions add on larger inactive groups onto the phase 1 product. The resulting metabolites have an increases molecular weight and are generally inactive, unlike phase 1 metabolites which often produce active metabolites. These conjugation reactions also yield metabolites that are easier to excrete than their initial form. There are four major mechanism of conjugation involved in the phase 2 reactions and they are: methylation, sulphation, acetylation and glucoronidation. The majority of the phase 2 metabolism is carried out in the smooth endoplasmic reticulum of liver cells, which small portions occurring in several other organs depending on the type of reaction. The liver is the main site of metabolism for several reasons; one being that it is the first organ to become perfused with drugs taken up from the gut, and compared to other organs there is a high concentration of drug metabolising enzymes.

Location of Metabolism: Liver, kidney, lung, CNS.
Enzymes Involved: Methyltransferase.
Methylation is a common pathway for drug metabolism but compared to the other reactions this process is considered a minor pathway. The goal of methylation is to make a drug slightly less water soluble, which would be done to drugs containing many polar groups. Methylation of the polar functional groups also essentially masks them from subsequent conjugation, possibly with a polar molecule such as glucoronic acid. Functional groups that are commonly methylated include but are not restricted to: phenols, catechols, amines, N-heterocyclics, sulfhydryl containing compounds and some metals1. Although this reaction is enzyme catalyzed, a cofactor known as SAM must also be present, this is shown below in figure 1.

Location of Metabolism: Liver, kidney, intestine.
Enzymes Involved: Sulfotransferases.
Another form of the phase 2 conjugation reactions is sulphation. This reaction type is not as common as the others and is mainly restricted to phenols, alcohols, arylamines and N-hydroxy compounds. Like several of the other reactions this one is enzyme catalyzed but requires a cofactor, 3’-phosphoadenosine 5’-phosphosulfate4 as the source of the sulphates. This method of phase 2 metabolism can form reactive metabolites, which is uncommon for phase 2 reactions. Primary amines, secondary amines, secondary alcohols and phenols react to form reactive sulphates which can act as toxic alkylating agents1.An example of sulphation is depicted below in figure 3.


Figure 3. Sulphation reaction example.
Location of Metabolism: Liver, lung, spleen, gastric mucosa, RBC’s, lymphocytes.
Enzymes Involved: N-acetyltransferases, Bile acid-CoA N-acyltransferases.
This is a major route for drug metabolism, especially for drugs containing aromatic amine and hydrazine groups (R-NH-NH-COCH3). Many of the functional groups utilized by the methylation reactions also work for acetylation1. Acetylation is the simple addition of an acetyl group to a molecule. This process involves the use of cofactors, such as acetyl coenzyme A (acetyl CoA), the structure of which is depicted below. The actual acetylating enzymes are mainly found in the liver but are distributed all over the body. This enzymes are known for their wide variability between species3.


Location of Metabolism: Liver, kidney, intestine, lung, skin, prostate, brain.
Enzymes Involved: UDP-glucuronosyltransferases.
This reaction is simply the conjugation of glucoronic acid and is the most common form of phase 2 reactions. Phenols, alcohols, hydroxylamines and carboxylic acids will form O-glucuronides using an enzyme and energy to attach glucoronic acid. The resulting metabolites are much more polar and much heavier because glucoronic acid has a molecular weight of approximately 194 AMUs. This allows for easier excretion via urine due to the presence of more polar functional groups; however if the resulting metabolite has an atomic weight greater than 300 AMU than the metabolite will be excreted via bile1. An example schematic of several reactions is shown below.

[1] G.L. Patrick. 2009. An Introduction to Medicinal Chemistry; Fourth Edition. Oxford University Press, Oxford.
[2] A. Rostami-Hodjegan and G. Tucker. 2007. Simulation and prediction of in vivo drug metabolism in human populations from in vitro data. Nature Reviews Drug Discovery 6 (2): 140–8.
[3] K. Sadoul, C. Boyault, M. Pabion, S. Khochbin. 2008. Regulation of protein turnover by acetyltransferases and deacetylases. Biochimie 90 (2): 306–12.
[4] Christopher Walsh. 2006. Chapter 5 - Protein Methylation; Posttranslational modification of proteins: expanding nature's inventory. Roberts and Co. Publishers.