Kinetic and Chemical Mechanisms of Yeast Formate Dehydrogenase

Yeast formate dehydrogenase has an ordered kinetic mechanism with NAD adding before formate. NAD analogues are substrates, but formate is the only molecule oxidized. Anions are competitive inhibitors vs. formate and bind only to E-NAD. Linear triatomic anions are the best inhibitors, with azide (K_i = 7 nM) showing tight binding inhibition behavior and appearing to be a transition-state analogue. pH profiles of the kinetic parameters show that a group in E-NAD with a pK of 8.3 must be protonated for binding of azide and formate. Since the pK is elevated to 9.8 upon formate binding, it is probably a cationic acid involved in substrate binding. A group with pK = 6.4 and no temperature dependence must be ionized for binding of azide and formate, and another group with pK = 5.9 must be ionized for catalysis, but the roles of these groups in the mechanism are not clear. A ¹³C isotope effect of 1.043 with either un-labeled or deuterated formate shows that formate has a very low commitment to catalysis, and all isotope effects observed are intrinsic ones on the chemical reaction. The deuterium isotope effect on V/K_formate varies with the nature of the nucleotide, presumably as the result of changes in transition-state structure. The value of 2.8 with NAD probably results from a late transition state, while the values of 4.4 for thio-NAD, 6.9 for acetylpyridine-NAD, and 3.8 for pyridinecarboxaldehyde-NAD represent progressively earlier transition states. By contrast, the ¹³C isotope effect drops slightly to 1.036 with acetylpyridine-NAD. These data are explained if both a higher redox potential (that is, more electrophilic C-4) for the nucleotide and an increased distance between formate and nucleotide in the transition state (indicated by a lower V_max, and increased activation energy for the reaction) cause the transition state to become earlier.