Three possible mechanisms for the reactions catalyzed by triosephosphate isomerase (TIM) have been studied by the combined quantum mechanical/molecular mechanical (QM/MM) approach at a number of QM levels including AM1, AM1 with specific reaction parameters (SRP), and B3LYP/6-31+G(d,p). The comparison of the various QM levels is used to verify the adequacy of our recent B3LYP/MM analysis of the reaction mechanism (Cui et al. J. Am. Chem. Soc. 2001, 123, 2284), which showed that the intramolecular proton transfer pathway is ruled out, due largely to the unfavorable interaction between the transition state and His 95. The relative contributions from the two other proposed pathways, however, are difficult to determine at the present level of theory; both pathways are also consistent with available experiments. To obtain information about the role of the enzyme, density functional calculations were made for model systems in the gas phase and in solution; selected models were also studied with ab initio calculations at the levels of MP2 and CCSD to confirm the B3LYP results. Mulliken population analysis of the transition states demonstrates that hydrogen transfers essentially as proton for all the reactions in TIM, with an electron population between +0.33 and +0.44. Adiabatic mapping calculations for path A indicate that the two relevant proton-transfer steps between the substrate and His 95 proceed in a nearly concerted manner. The QM model calculations in solution and a QM/MM perturbation analysis shows that a number of factors combine to yield the rate enhancement by a factor of 10(9) in TIM. These include orienting catalytic groups (e.g., Glu 165, His 95) in good positions for the proton transfers, employing charged and polar groups (e.g., Lys 12, Asn 10) that stabilize the reaction intermediates and permitting flexibility of the catalytic groups (e.g., Glu 165 along path C). Some residues far from the active site, such as the main-chain atoms in Gly 210, as well as certain water molecules, also make significant contributions. For the electrostatic interaction and polarization to function effectively, the active site of TIM has a relatively low effective dielectric "constant", which reflects the structural integrity of the enzyme active site as compared with solution. Short hydrogen bonds occur during the reaction (e.g., between the reactant substrate and Glu 165), but the calculated energetics indicate that they do not have a specific role in catalysis; i.e., no contribution was found from the rather short hydrogen bond between His 95 and the substrate in path A.