Mammalian 3-oxosteroid delta4-delta5-isomerase. A membrane-bound enzyme. II. Activation by divalent cations.

Alkaline-earth ions (Mg2+, Ca2+ and Sr2+) have two specific effects on the kinetic parameters of the beef adrenal 3-oxosteroid delta4-delta5-isomerase activity in the microsomes and in the particles obtained after disrupting the membrane structure by action of 1 M MgCl2. On the microsomal enzyme, a 2-fold increase of V is observed with the three cations under study. The small difference in the effect of the three ions could be related to their hydration energy. It is suggested that the interaction of the ion with water is the determinant step of the activation mechanism and not the fixation of the ion on the enzyme or on some others possible binding sites in this system. With the enzyme in the proteolipidic particles, the use of EDTA as a chelating agent for the cations present in the enzymatic assay, allows the characterization of two effects: at low concentration of EDTA, an increase of Km is observed and at higher concentration (2 mM), V is decreased. A subsequent addition of Mg2+ leads to an activation in two steps: V is increased in the first step without change in Km, the second step consists of a decrease of Km without any change in V. A relation between the structural perturbations induced by the ions (Gallay, J., Vincent, M. and Alfsen, A. (1975) Biochim. Biophys. Acta 397, 489-500) and their kinetic effect on the enzymatic reaction is established.