Kinetic isotope effect and the presteady-state kinetics of the reaction catalyzed by the bacterial formate dehydrogenase.
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 HVm/DVm and H(Vm/KM)/D(Vm/KM) 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 (kon) and dissociation (koff) at the stages of the binary--ternary enzyme-substrate complexes for the random equilibrium 2-substrate kinetic mechanism. The kon and koff values obtained for the bacterial formate dehydrogenase by using the proposed approach for NAD+ were (4.8 +/- 0.8)*10(5)M-1s-1 and (90 +/- 10) s-1, and for formate (2.0 +/- 1.0)*10(4) M-1s-1 and (60 +/- 20) s-1, respectively.
Tishkov
V I
VI
A.N. Bach Institute of Biochemistry, USSR Academy of Sciences, Moscow.
Galkin
A G
AG
Egorov
A M
AM
D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D003903:Deuterium; D005560:Formate Dehydrogenases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008956:Models, Chemical; D011549:Pseudomonas / Q000201:enzymology*
D003903:Deuterium; D005560:Formate Dehydrogenases; D000445:Aldehyde Oxidoreductases