Properties of the Nicotinamide Adenine Dinucleotide-binding Sites of L-α-Glycerophosphate Dehydrogenase

Abstract

Abstract l-α-Glycerophosphate dehydrogenase was demonstrated to be inhibited by a homologous series of N1-alkylnicotinamide chlorides, ranging from the N1-methyl to the N1-dodecyl derivative, inclusive. The inhibition obtained was observed to be reversible and competitive with respect to the coenzyme NAD. The binding of N1-alkylnicotinamide chlorides larger than the N1-pentyl derivative to glycerophosphate dehydrogenase was shown to be facilitated through nonpolar interactions with the enzyme. Adenosine, adenylic acid, adenosine diphosphate, and adenosine diphosphoribose were also shown to be coenzyme-competitive inhibitors of glycerophosphate dehydrogenase. The effectiveness of binding of these compounds increased with the size of the adenine derivative employed. Multiple inhibition analysis was used to demonstrate simultaneous binding of inhibitors that interact with different regions of the NAD-binding site of the enzyme. Inhibitors interacting with the same regions of the NAD-binding site were shown to mutually exclude one another from binding to the enzyme. It was suggested that the binding of NAD to glycerophosphate dehydrogenase occurs through interactions involving the adenosine moiety, the pyrophosphate grouping, and the positively charged nicotinamide ring system. The presence of a hydrophobic region near the NAD-binding site of the enzyme was indicated.