Kinetic isotope effect and the presteady-state kinetics of the reaction catalyzed by the bacterial formate dehydrogenase

Abstract

The primary kinetic isotope effect of the reaction catalyzed by NAD +-dependent formate dehydrogenase (EC 1.2.1.2) from the methylotrophic bacterium Pseudomonas sp. 101 has been studied. Analysis of the ratios HV m/ DV m and H(V m/K M)/ D(V m/K M) in the pH range 6.1–7.9 showed that the transfer of hydride ion in ternary enzyme-substrate complex is a limiting step of the reaction, and the formate binding to the binary complex (formate dehydrogenase + NAD +) reached equilibrium when the pH of the medium was increased. An approach has been developed to determine the elementary constants of substrate association ( k on ) and dissociation ( k off ) at the stages of the binary-ternary enzyme-substrate complexes for the random equilibrium 2-substrate kinetic mechanism. The k on and k off values obtained for the bacterial formate dehydrogenase by using the proposed approach for NAD-were (4.8 ± 0.8) ∗ 10 5 M −1 s −1 and (90 ± 10) s −1 , and for formate (2.0 ± 1.0) ∗ 10 4 M −1 s −1 and (60 ± 20) s −1 , respectively.