Inhibition of β-hydroxysteroid dehydrogenase II. Kinetics and pH effect

Abstract 1. 1. An investigation was conducted on the kinetics of inhibition of β-hydroxysteroid dehydrogenase (3(or 17)-β-hydroxysteroid: NAD(P) oxidoreductase, EC 1.1.1.51) from Pseudomonas testosteroni . The natural estrogen, estradiol-17β ( K i = 1·10 −5 M) was a non-competitive inhibitor as were 2-hydroxymethylene-17α-methylandrostan-17β-ol-3-one ( K i = 3·10 −7 M) and 2α-cyano-4,4,17α-trimethyl-androst-5-en-17β-ol-3-one ( K i = 7·10 −7 M). 4,4-Dimethyl-17β-hydroxyandrost-5-eno[3,2-c]pyrazole ( K i = 5·10 −7 M) and 17β-hydroxy-4,4,17a-trimethylandrost-5-eno[2,3-d]isoxazole ( K i = 4·10 −6 M) were competitive inhibitors. These four synthetic androstane or androstene derivatives were more tightly bound to the enzyme than was estradiol-17β. 2. 2. The degree of inhibition by estradiol-1gb and by diethylstilbestrol was found to vary with pH. Both estrogenic compounds were between 4 and 20 times more effective inhibitors when the pH was made 4 units more alkaline. Possible reasons for this behavior are a change in the degree of ionization of groups at or near the active site(s) of the enzyme and a contribution of phenolate ion by these estrogenic compounds.