Kinetic study of the enzymatic cycling reaction conducted with 3alpha-hydroxysteroid dehydrogenase in the presence of excessive thio-NAD(+) and NADH

We have established a simple kinetic model applicable to the enzyme cycling reaction for the determination of 3alpha-hydroxysteroids. This reaction was conducted under the reversible catalytic function of a single 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) with nucleotide cofactors, thio-NAD(+) (one of the NAD(+) analogues) for the oxidation of 3alpha-hydroxysteroids and NADH for the reduction of 3-oxosteroids. This model was constructed based on the reaction mechanism of 3alpha-HSD, following an ordered bi-bi mechanism with cofactor binding first, under the assumption that the respective enzyme-cofactor complexes were distributed according to the initial ratio of thio-NAD(+) to NADH by the rapid equilibrium of both enzyme-cofactor complexes. The cycling rate in the new kinetic model could be expressed with the dissociation constants of enzyme-cofactor complexes and the initial concentrations of cofactors and enzyme. The cycling rate was verified by a comparison with the experimental data using 3alpha-HSD from Pseudomonas sp. B-0831. The results showed that the experimental data corresponded well with the results obtained from the kinetic model.