Cytosine thiolation

Other reactions characterized for pyrimidine residues include mercuration at C-5 of cytosine or uracil (Hopman et al., 1986), cycloaddition to the 5,6-double bond of thymine and uracil (Cimino et al., 1985), and thiolation at the C-4 amino group of cytosine (Malcom and Nicolas, 1984). 

The catalytic mechanism of the pyrimidine-5 methylation in nucleic acids is more complex as it involves covalent catalysis. The mechanism is common for numerous DNA/RNA cytosine and uracil MTases as well as for thymidylate synthase (although the latter uses tetrahydrofolic acid as the methyl donor) and has been studied in detail in several systems (50). Here, the cytosine-5 methylation in DNA is presented as an example (see Fig. 4a). The C5-position of cytosine, which is part of an aromatic ring, does not carry sufficient nucleophilicity for a direct methyl group transfer. The continuity of the aromatic system is disrupted by a nucleophilic attack of thiolate (from a conserved... 

Figure 4 Catalytic activation of cytosine for C5-methylation by nucleophilic addition of a thiolate at the C6 position, (a) The chemical mechanism of enzymatic DNA cytosine-S methylation. Mechanism-based inhibition of DMA MTases by cytidine analogs 5-fluoro-2 -deoxycytidine (b), 5-aza-2 -deoxy-cytidine (c), and 2-pyrimidinone-l-p-D-(2 -deoxyriboside) (d).

Figure 13.10 Proposed mechanism for 5-methylation of cytosine residue in DNA. Reaction sequences catalyzed by H. haemolyticus methyltransferase are depicted. The formation of the methylated DNA intermediate involving thiolate of Cys81 is shown...

C-methylation reaction is the methylation of the C-5 position of cytosine in DNA. In this case, the carbon C-5 of cytosine cannot directly act as a nucleophile. The electron withdrawal by N-3 and the carbonyl, however, makes the C-5—C-6 double bond electron deficient and prone to attack by nucleophiles in a reaction that is similar to a Michael reaction. In DNA methyltransferases (DNMTs), this nucleophile is the thio-late from a Cys residue. The addition product is nucleophilic and reacts with SAM via an Si,j2-like mechanism to capture the methyl group. The resulting intermediate then eliminates the Cys of DNMT to give the methylated cytosine product (Figure 1.9). The methylation of C-5 of cytosine is an example of converting an electron-deficient methyl acceptor to a nucleophile for the methyl-transfer reaction by addition of an active site Cys thiolate. 

---