Kinetic studies on bovine cytochrome P45011β catalyzing successive reactions from deoxycorticosterone to aldosterone

The reactions for the synthesis of aldosterone from deoxycorticosterone were investigated kinetically in the membrane-reconstituted system with bovine cytochrome P450(11 beta) at 37 degrees C, Reaction rapid-quenching experiments for the metabolism of deoxycorticosterone by P450(11 beta) showed that aldosterone was produced via corticosterone, not via 18-hydroxydeoxycorticosterone. The kinetic analysis revealed that aldosterone was formed successively from fractions of intermediate metabolites which did not dissociate from P450(11 beta). The rate of each reaction step in the successive reactions was estimated from the combination of results of the rapid-quenching experiments and the metabolism of deoxycorticosterone in the presence of an excess amount of substrate, in which the dissociation of final product, aldosterone, from the enzyme was the slowest step in the synthesis from deoxycorticosterone. Under steady-state reaction conditions, the interaction of P450(11 beta) with P450(scc) stimulates the production of corticosterone from deoxycorticosterone by about 10-fold but inhibits further reactions from corticosterone. The rapid-quenching experiments showed, however, that the rate constant for the 11 beta-hydroxylation of deoxycorticosterone for corticosterone production in the presence of P450(scc) was almost the same as that without P450(scc). The interaction of P450(11 beta) with P450(scc) in the reaction system for deoxycorticosterone metabolism was found to slow the rate of the subsequent Is-hydroxylation of the produced corticosterone and to accelerate the dissociation of the corticosterone from P450(11 beta), which stimulated the corticosterone production and inhibited the further reaction for aldosterone synthesis in the steady-state reaction.