In previous report, with the aid of receptor-oriented pharmacophore-based in silico screening, we characterized three novel hPNMT inhibitors (YPN010, YPN016, and YPN017) and proposed that the hydrogen bonding interaction between inhibitors and side chain of Lys57 is very important to inhibitory activity of hPNMT. To confirm the importance of Lys57, mutant with substitution of Lys57 with Ala was cloned and binding study was performed for a K57A mutant of hPNMT using STD-NMR and fluorescence experiments. The binding constants for three novel inhibitors with mutant hPNMT were dramatically decreased compared to those with wild-type protein. K57A mutant-induced conversion of noradrenaline to adrenaline was suppressed about 95 % compared to wild-type hPNMT. Mutagenic analysis using a K57A mutant confirmed the importance of the Lys57 residue in binding of the inhibitor candidate to hPNMT as well as enzymatic activity of hPNMT, implying that these results are consistent with our binding model.