Combined coenzyme-substrate analog of various dehydrogenases. Synthesis of (3S)- and (3R)-5-(3-carboxy-3-hydroxypropyl)nicotinamide adenine dinucleotide and their interaction with (S)- and (R)-lactate-specific dehydrogenases

Abstract

Two diastereomeric NAD+ derivatives were synthesized in which the substrates of (S)- and (R)-lactate-specific dehydrogenases are covalently attached via a methylene spacer at position 5 of the nicotinamide ring. The corresponding nicotinamide derivatives were obtained stereospecifically by enzymatic reduction of 5-(2-oxalylethyl)nicotinamide. (3S)-5-(3-Carboxy-3-hydroxypropyl)-NAD+ undergoes an intramolecular hydride transfer in the presence of pig heart lactate dehydrogenase, forming the corresponding coenzyme-substrate analog composed of pyruvate and NADH. No cross-reaction products resulting from an intermolecular reaction are observed. Two [Lactobacillus leichmannii and Limulus](R)-lactate specific dehydrogenases [EC 1.1.1.28], however, do not catalyze a similar reaction in either 1 of the 2 diastereomers. A possible arrangement of the substrates in the active centers of these enzymes is proposed. 5-Methyl-NAD+ and 5-methyl-NADH are active coenzymes of pig heart lactate dehydrogenase in contrast to reports in the literature. (S)-Lactate binds to this enzyme in the absence of coenzyme, exhibiting a dissociation constant of 11 mM.