Open Access3α‐Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni: Kinetic Properties with NAD and Its Thionicotinamide Analogue
Author(s) -
SKÅLHEGG Bjørn A.
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb09901.x
Subject(s) - nad+ kinase , cofactor , oxidoreductase , dehydrogenase , chemistry , stereochemistry , enzyme , hydroxysteroid dehydrogenases , hydroxysteroid dehydrogenase , substrate (aquarium) , biochemistry , biology , ecology
The kinetics of 3α‐hydroxysteroid: NAD oxidoreductase (EC 1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) have been investigated. The kinetic analysis based on initial activity measurements and product inhibition studies, indicates that the addition of substrate to the enzyme and the release of products from it, follows an obligatory order (ordered bi bi mechanism). The ability of the enzyme to utilize the thionicotinamide analogue of NAD (sNAD) as cofactor has been investigated using various 3α‐hydroxysteroids from both the C 19 , C 21 and C 24 series. The results show that the reaction velocity with sNAD as the cofactor is generally lower than with NAD. The decrease, however, varies considerably, being negligible with some steroids such as litocholic acid and deoxycholic acid and very pronounced with other such as tetrahydrocortisol and tetrahydrocortisone. The introduction of an 11β‐hydroxy or an 11‐oxo group into the steroid molecule significantly reduces the ability of the enzyme to attack the 3α‐hydroxy group. No such effect could be seen when the 11‐hydroxy group was in the α‐position. The results also indicate that, whereas NAD can serve as cofactor for both the monomeric and the dimeric forms of the enzyme, sNAD only acts as cofactor for the monomeric form. Thus sNAD is a valuable tool for the study of the reversible, concentration‐dependent monomeric‐dimeric transition of the 3α‐hydroxysteroid dehydrogenase.