We have performed long-term molecular dynamics simulations of pyruvate decarboxylase from Zymomonas mobilis. Nine structures were modeled to investigate mechanistic questions related to binding of the cofactor, thiamin diphosphate (ThDP), and the substrate in the active site. The simulations reveal that the proposed three ThDP-tautomers all can bind in the active site and indicate that the equilibrium is shifted toward 4'-aminopyrimidine ThDP in the absence of substrate. 4'-Aminopyrimidinium ThDP is found to be a likely intermediate in the equilibrium. Mutations of important active site residues, Glu473Ala and Glu50Ala, were modeled to further elucidate their catalytic role. Formation of the catalytic important ylide by deprotonation of ThDP(C2) is investigated. Only the less favored tautomer, 1',4'-iminopyrimidine ThDP (imino-ThDP), could be deprotonated. The two other tautomers of ThDP could not be activated at the C2-position, thus, explaining the mechanistic importance of the less stable imino-ThDP. Finally, binding of pyruvate in the active site with the cofactor modeled as the nucleophilic ylide (ylide-ThDP) is studied. The carbonyl group of the substrate forms a hydrogen bond to Tyr290(OH). No hydrogen bond could be identified between ThDP(N4') and the substrate. The geometry of the substrate binding is well-suited for a nucleophilic attack by ylide-ThDP(C2). We propose that a proton relay from His113 via Asp27 and Tyr290 to the carbonyl oxygen atom of the substrate may be involved in the mechanism.