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.
Department of Diagnostics Research and Development, Division of Fine Chemicals and Diagnostics, Asahi Kasei Pharma Corporation, 632-1, Mifuku, Ohito-cho, Shizuoka 410-2321, Japan.
Shigeru
S
Ueda
Masayuki
M
Oda
Shigeyuki
S
Imamura
Masatake
M
Ohnishi
D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific) / Q000378:metabolism*; D003627:Data Interpretation, Statistical; D007700:Kinetics; D008954:Models, Biological; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism*
D009243:NAD; C003451:thionicotinamide adenine dinucleotide; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)