An investigation of the active site of lactate dehydrogenase with NAD+ analogues.

The kinetic properties of 18 NAD+ analogues, with alterations to the nicotinamide moiety, have been studied with respect to dogfish, M4, rabbit M4 and beef H4 lactate dehydrogenase. The size of the groups present at C-3 of the pyridinium can be increased quite extensively without loss of coenzyme activity. Groups tested were thioamide, methyl, ethyl, diazoketone and chloroacetyl. Substitutions at positions C-4 and C-5 prevent proper positioning for hydride transfer and can hinder binding to the enzyme. The kinetic properties of pyridine-adenine dinucleotide and its 3-iodo derivative reveal the binding role of the amide at C-3 whereas 3-cyanopyridine-adenine dinucleotide is a strong inhibitor.