THEORETICAL INVESTIGATION OF THE HYDRIDE TRANSFER FROM FORMATE TO NAD(+) AND THE IMPLICATIONS FOR THE CATALYTIC MECHANISM OF FORMATE DEHYDROGENASE

The hydride transfer reaction between formate and NAD(+) has been inve stigated by using molecular orbital theory in combination with continu um solvation models. The reaction in the gas phase is extremely exothe rmic due to the instability of the charged reactant species. The calcu lations reveal that during the hydride transfer the pyridine ring of N AD(+) takes a quasi-boat conformation. The nitrogen atom of the pyridi ne ring remains planar, which is in agreement with the experimentally established N-15 kinetic isotope effect (1.004 +/- 0.001) of the forma te dehydrogenase catalyzed oxidation of formate to carbon dioxide. The computed value at the HF/6-31+G(d,p) level of theory for the N-15 kin etic isotope effect is 1.0042. In solution, however, there is a potent ial energy barrier for the hydride transfer. At the MP2/6-31G(d)//HF/6 -31G(d) level of theory the self-consistent reaction field approach gi ves a barrier height of 9.0 kcal/mol in acetonitrile (epsilon = 35.9). Direct nucleophilic addition of one of the carboxylate oxygens of for mate to the pyridine ring of NAD(+) competes with hydride transfer, an d this study reveals that this nucleophilic addition is likely to be p referred over the hydride transfer in the gas phase. Thus, the NAD(+)- dependent formate dehydrogenase must orient the substrate formate in t he active site in such a fashion as to prevent this competing reaction from occurring. According to the recently solved X-ray crystal struct ure, it is clear that the Arg-284 and Asn-146 are the two critical ami no acid residues that hold formate in the productive orientation for h ydride transfer.