We found that the histidine chemical modification of tyrosinase conspicuously inactivated enzyme activity. The substrate reactions with diethylpyridinecarbamate showed slow-binding inhibition kinetics (KI = 0.24 +/- 0.03 mM). Bromoacetate, as another histidine modifier, was also applied in order to study inhibition kinetics. The bromoacetate directly induced the exposures of hydrophobic surfaces following by complete inactivation via ligand binding. For further insights, we predicted the 3D structure of tyrosinase and simulated the docking between tyrosinase and diethylpyridinecarbamate. The docking simulation was shown to the significant binding energy scores (-3.77 kcal/mol by AutoDock4 and -25.26 kcal/mol by Dock6). The computational prediction was informative to elucidate the role of free histidine residues at the active site, which are related to substrate accessibility during tyrosinase catalysis.