The physical nature of interactions within the active site of cytosine-5-methyltransferase (CMT) was studied using a variation-perturbation energy decomposition scheme defining a sequence of approximate intermolecular interaction energy models. These models have been used to analyze the catalytic activity of residues constituting cytosine-5-methyltransferase active site as well their role in the binding group of de novo designed inhibitors. Our results indicate that Glu119, Arg163, and Arg165 appear to play the dominant role in stabilizing the protonated transition state structure and their influence can be qualitatively approximated by electrostatic interactions alone. The stabilization of neutral structures of the alternative reaction pathway is small, which might suggest the protonated pathway as preferred by the enzyme. Exchange and delocalization terms are negligible in most cases, or they cancel each other to some extent. Interactions of inhibitors with the CMT active site are dominated by electrostatic multipole contributions in analogy with previously studied transition state analogue inhibitors of leucyl aminopeptidase.