Interaction of nicotinamide adenine dinucleotide with 20.BETA.-hydroxysteroid dehydrogenase from Streptomyces hydrogenans.

Abstract

Some information about the binding of coenzyme to 20β-hydroxysteroid dehydrogenase [EC 1.1.1.53] from Streptomyces hydrogenans was obtained from the coenzyme specificity and from inhibition studies using various nucleotides. Nicotinamide adenine dinucleotide (NAD) and its analogs served as coenzymes of the enzyme, but NADP did not. When NAD was modified in the adenine ring, the apparent Km values increased considerably and these values were very high as compared with those of the NAD analogs modified in the nicotinamide moiety. Adenosine 5'-diphosphate (ADP)-ribose, ADP and 5'-AMP were strong competitive inhibitors with respect to coenzyme. ATP and adenosine moderately inhibited the enzyme activity. IMP, IDP, IDP-ribose, 1, N6-etheno-adenosine 5'-monophosphate, 2'-AMP and 3'-AMP were very poor inhibitors. The N6-amino group of the adenine moiety may be essential for the binding of the coenzyme to the enzyme, and the phosphate group at the 5'-position of ribose of the adenosine moiety may also play an important role in the binding process. The presence of a phosphate group at the 2'-or 3'-position of ribose of the adenosine moiety resulted in a significant decrease in the binding capacity of adenine nucleotides to the enzyme. 2'-Deoxy-5'-AMP and 3'-deoxy-5'-AMP were very poor inhibitors relative to 5'AMP, indicating that there may be an interaction between the 2'-or 3'-hydroxyl group and the coenzyme binding site. Nicotinamide and NMN showed only slight inhibitory action compared with adenine nucleotides. It was suggested that the adenosine moiety of the coenzyme may have an important role in the binding interaction with the enzyme.