Formate dehydrogenase (FDH) from Pseudomonas sp. 101 is a homodimeric enzyme that catalyzes oxidation of formate and the concomitant reduction of NAD(+) to produce NADH and CO2. The dynamic motions and distances between functional groups in the active site of the formate dehydrogenase including the NAD+ cofactor and substrate have been investigated by molecular dynamics (MD) simulations, incorporating the substrate in one subunit (E.S) and the transition state in the other subunit (E.TS). Experimental kinetic isotope effects and calculated isotope effects are in excellent agreement, thus, the transition state in the enzymatic reaction is expected to closely resemble the structure determined by ab initio calculations. The simulation shows that the formate is held in place by persistent electrostatic interactions consisting of a bifurcated hydrogen bond between one formate oxygen and the guanido hydrogens of Arg284, and single hydrogen bonds from the other formate oxygen to a side chain amide proton of Asn146 and the backbone amide proton of Ile122. The conserved residues Arg284, Asn146; and Ile122 serve as pivots about which the C-H of formate swings to and from the C4N of NAD(+). The C4N of NAD(+) and the formate hydrogen are in position to react (near attack conformations, NACs) approximately 1.5% of the simulation time. An additional effect of the hydrogen bonding of the formate oxygens to Arg284, Asn 146, and lie 122 is to prevent nucleophilic attack of the carboxylate on NAD(+) to form an ester, which is the reaction favored in the gas phase. His332 plays a role in both the binding of formate and the generation of the near attack conformations. Further insight into the roles of other conserved amino acids (Pro97, Phe98, Asp308, and Ser334) at the catalytic site is provided. Comparisons of the electrostatic interactions at the active site of the enzyme with substrate and transition state show changes in hydrogen bonding due to charge differences; however, these changes are not consistent with the hypothesis of preferential stabilization of the transition state over the ground state.