Role of thiol group(s) in the enzymatic activity of the membrane-bound [formula omitted] dehydrogenase from bovine adrenal cortex microsomes

Abstract

The effect of different sizes of thiol groups reagents on the kinetic parameters of the membrane-bound Δ 5-3β- hydroxy-steroid dehydrogenase (3(or 17)-β-hydroxysteroid:NAD(P) + oxidoreductase, EC 1.1.1.51) from bovine adrenal cortex microsomes was examined. Iodoacetamide and parachloromercuribenzoate increased the maximum velocity 1.6 times and also the K m for adrost-5-en-3β-ol-17-one by factors of 1.5 and 5.5, respectively. Hydrophobic reagents of larger size led to a large decrease of the maximum velocity and further increase in the K m for adrost-5-en-3β-ol-17-one. Androst-5-en-3β-ol-17-one hindered the activating effect of iodoacetamide. Similar results were found with the NAD + analogue, 3-chloro-acetyl pyridinium adenine dinucleotide, an electrophilic reagent. A decrease of the maximum velocity was observed (38% inhibition at the highest concentration used). As with other SH reagents, the K m for androst-5-en-3β-ol-17-one was increased 5 times. Preincubation of membranes with iodoacetamide prevented the enzyme from inhibition by 3-chloroacetyl pyridinium adenine dinucleotide. Protection experiments with androst-5-en-3β-ol-17-one, oestr-1,3,5(10)6,8-pentaen-3-ol-17-one and NAD + demonstrated that the steroids were more effective than NAD + in preventing the enzyme from inhibition. In all cases, the K m for NAD + was unaltered. From these results, it is suggested that thiol reagents, including 3-chloroacetyl pyridium adenine dinucleotide, substitute an SH group in the vicinity of the steroid-binding site of the enzyme.