Bull Semen Nicotinamide Adenine Dinucleotide Nucleosidase: III. PROPERTIES OF THE SUBSTRATE BINDING SITE

Abstract

Abstract The properties of the substrate binding site of the bull semen nicotinamide adenine dinucleotide nucleosidase were studied with a titrimetric assay. The adenosine derivatives, adenosine, AMP, ADP, and ADP-ribose, were found to inhibit the NADase-catalyzed hydrolysis of NAD. Inhibition by these compounds was observed to be competitive with respect to NAD. The importance of an adenosine region and a pyrophosphate region at the substrate binding site of this enzyme was demonstrated by the relative Ki values obtained for these compounds. N1-Alkylnicotinamide chlorides were also shown to be substrate-competitive inhibitors of the NADase-catalyzed hydrolysis of NAD. These compounds were suggested to be bound at a pyridinium ring region of the substrate binding site. The positive chain length effect observed in the binding of N1-alkylnicotinamide chlorides, N1-ethyl- to N1-dodecylnicotinamide chloride, inclusive, to the enzyme suggested the presence of a hydrophobic region close by the pyridinium ring region. Multiple inhibition studies demonstrating simultaneous binding of adenosine derivatives and N1-methylnicotinamide chloride indicate that the binding of these inhibitors occurs at different parts of the substrate binding site.