DNA (Deoxyribonucleic acid) methylation is a process by which methyl groups are transferred to the DNA molecule using S-adenosyl methionine (SAM) as the methyl donor. Enzymes used to catalyze this transfer are DNA methyltransferase (DNA MTase).
Mechanism based inhibitors are the competitive inhibitors that have structural similarity to the substrate or the product of the target enzyme. This allows the formation of a stable inhibitor enzyme complex with the help of covalent bonding.
Inhibitors can incorporate into the DNA only in their tri-phosphorylated active forms during DNA replication in the S phase (Synthesis- phase) of the cell cycle. The enzyme used the replication is the DNA polymerase. 5-Azacytidine, a ribose nucleoside is chemically modified to a deoxyribonucleoside triphosphate that is 5-aza-2′-deoxycytidine-triphosphate.
On its incorporation into the DNA the normal cytosine is replaced. The DNA methyl-transferases are responsible for recognition of Azacytosine-guanine dinucleotides that are able to catalyze the methylation reaction with nucleophilic attack. As the result of the reaction, the covalent bond formation takes place between carbon-6 (C-6) of the cytosine ring and the enzyme. Hence, a stable intermediate is formed.
The proton attack at carbon-5 (C-5) not occur because C-5 in azacytosine is substituted by nitrogen that lacks protons. This results in the irreversible formation of a covalent complex. Thus, inhibitor traps the enzyme on the DNA, resulting in the degradation of the enzyme.
5-fluorodeoxycytidine follows similar reaction. It has a fluorine atom instead of the proton at C-5 resulting in the formation of a stable covalent complex trapping the enzyme leading to its degradation.