| Abstract
| - Formate dehydrogenase (FDH) from Pseudomonas sp. 101 is a homodimeric enzyme that catalyzesoxidation of formate and the concomitant reduction of NAD+ to produce NADH and CO2. The dynamic motionsand 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 thesubstrate in one subunit (E·S) and the transition state in the other subunit (E·TS). Experimental kinetic isotopeeffects and calculated isotope effects are in excellent agreement, thus, the transition state in the enzymaticreaction is expected to closely resemble the structure determined by ab initio calculations. The simulationshows that the formate is held in place by persistent electrostatic interactions consisting of a bifurcated hydrogenbond between one formate oxygen and the guanido hydrogens of Arg284, and single hydrogen bonds from theother formate oxygen to a side chain amide proton of Asn146 and the backbone amide proton of Ile122. Theconserved residues Arg284, Asn146, and Ile122 serve as pivots about which the C−H of formate swings toand from the C4N of NAD+. The C4N of NAD+ and the formate hydrogen are in position to react (near attackconformations, NACs) approximately 1.5% of the simulation time. An additional effect of the hydrogen bondingof the formate oxygens to Arg284, Asn146, and Ile122 is to prevent nucleophilic attack of the carboxylate onNAD+ to form an ester, which is the reaction favored in the gas phase. His332 plays a role in both the bindingof formate and the generation of the near attack conformations. Further insight into the roles of other conservedamino acids (Pro97, Phe98, Asp308, and Ser334) at the catalytic site is provided. Comparisons of the electrostaticinteractions at the active site of the enzyme with substrate and transition state show changes in hydrogenbonding due to charge differences; however, these changes are not consistent with the hypothesis of preferentialstabilization of the transition state over the ground state.
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