Human 8-oxoguanine-DNA glycosylase (hOGG1) possesses very high specificity for 8-oxoguanine (oxoG), even though this damaged base differs from normal guanine by only two atoms. Our aim was to determine the roles of certain catalytically important amino acid residues in the hOGG1 enzymatic pathway and describe their involvement in the mechanism of DNA lesion recognition. Molecular dynamic simulation and pre-steady-state fluorescence kinetics were performed to analyze the conformational behavior of wild-type hOGG1 and mutants G42S, D268A, and K249Q as well as damaged and undamaged DNA. A loss of electrostatic interactions in the K249Q mutant leads to the disruption of specific contacts in the active site of the enzyme and the loss of catalytic activity. The absence of residue Asp-268 abrogates the ability of the enzyme to fully flip out the oxoG base from the double helix, thereby disrupting proper positioning of the damaged base in the active site. Furthermore, substitution of Gly-42 with Ser, which forms a damage-specific H-bond with the N7 atom of the oxoG base, creates a stable H-bond between N7 of undamaged G and O gamma of Ser-42. Nevertheless, positioning of the undamaged base in the active site is unsuitable for catalytic hydrolysis of the N-glycosidic bond.