The existence and magnitude of effects of adenine nucleotides on the catalytic activity and the structure of yeast glyceraldehyde 3-phosphate dehydrogenase were evaluated for their significance to metabolic controls. The results also led to an elucidation of the principal features of the mechanism of binding of nicotinamide-adenine dinucleotide to the enzyme. The two nucleotide moieties in nicotinamideadenine dinucleotide were found to have almost totally separate functions, the adenine nucleotide moiety being mainly responsible for binding, and the nicotinamide nucleotide moiety for catalysis. Thus, glyceraldehyde 3-phosphate dehydrogenase was inhibited by the following compounds (Ki values in parentheses): adenine (10.2 mM), adenosine (2.1 mM), adenosine 5′-monophosphate (1.1 mM), adenosine diphosphate (1.5 mM), adenosine triphosphate (2.5 mM), adenosine 3′,5′-cyclic monophosphate (0.11 mM), and pyridine 3-aldehyde nicotinamide-adenine dinucleotide (0.27 mM). In each case, the inhibition was competitive with respect to nicotinamide-adenine dinucleotide (Km = 0.18 mM). Glyceraldehyde 3-phosphate dehydrogenase was not inhibited by nicotinamide mononucleotide. Therefore, the adenine nucleotide moiety was bound, and was essential for binding, while the nicotinamide was neither. The 6-amino group of the adenine moiety and 2′-hydroxyl group of the adenine-linked ribose moiety were essential for binding. A 5′-phosphate linked to adenosine greatly aided the binding, if it was a diester. The inhibition was fairly specific for purines, especially for adenine-containing compounds. Thus, the compounds 2′-deoxyadenosine, 2′-deoxyadenosine 5′-monophosphate, 2′-deoxyadenosine 5-triphosphate, adenosine 2-monophosphate, adenosine 3-monophosphate, adenosine 2,3-cyclic monophosphate, nicotinamide-adenine dinucleotide phosphate, inosine, inosine 5′-monophosphate, nicotinamide-6-deaminoadenine dinucleotide, nicotinamide mononucleotide, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate gave little or no inhibition. The adenosine 3,5-cyclic monophosphate was an extremely potent inhibitor and was bound to the enzyme even more tightly than nicotinamide-adenine dinucleotide. The inhibition of glyceraldehyde 3-phosphate dehydrogenase by adenosine 3,5-cyclic monophosphate, adenosine triphosphate, and other adenine-containing compounds merits consideration as a mechanism for control of glyceraldehyde 3-phosphate dehydrogenase activity. Binding of adenine-containing compounds may also be important in control of the level of glyceraldehyde 3-phosphate dehydrogenase. © 1969, American Chemical Society. All rights reserved.
