Synthesis and Kinetic Properties of a New NAD+ Derivative Carrying a Vinyl Group

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Abstract
A method for the synthesis of NAD+‐N6‐[N‐(N‐acryloyl‐1‐methoxycarbonyl‐5‐aminopentyl)‐propioamide] (monomeric NAD+ derivative), a new NAD+ derivative carrying a vinyl group, was described. By copolymerization with acrylamide 90% of the NAD+ derivative was converted to a water‐soluble macromolecular NAD+ derivative (polymeric NAD+ derivative). Both NAD+ derivatives were reduced completely with yeast alcohol dehydrogenase.High cofactor activities relative to free NAD+ and NADH were obtained for the monomeric derivatives of NAD+ (71–86%) and NADH (66–87%) with yeast alcohol, horse liver alcohol, lactate, and malate dehydrogenases. Lower but substantial relative cofactor activities were obtained for the polymeric derivatives of NAD+ (18–33%) and NADH (28–60%) with yeast alcohol, horse liver alcohol, and malate dehydrogenases. But lactate dehydrogenase had negligible activity both for the polymeric NAD+ and NADH derivatives.Kinetic studies were carried out with yeast alcohol dehydrogenase and lactate dehydrogenase, and the following kinetic constants were determined: the maximum velocity (V), the limiting Michaelis constants for coenzyme (Ka) and for substrate (Kb), and the dissociation constant of the enzyme‐coenzyme complex (Kia). In the reaction system with the monomeric NAD+ derivative and yeast alcohol dehydrogenase, the Kia value was similar to that for NAD+ and the values of V, Ka, and Kb were 38, 30, 21% of those for NAD+. In the reaction system with the polymeric NAD+ derivative and yeast alcohol dehydrogenase, the values of V, Ka, Kb, and Kia were 0.5, 4, 3, 3 times larger than those for the monomer. In the reaction system with the monomeric NAD+ derivative and lactate dehydrogenase, the values of V, Ka, Kb and Kia were 39, 16, 36, 57% of those for NAD+. From these results the following conclusions were obtained: (a) the modification of NAD+ by alkylating at position 6 in the adenine moiety of NAD+ caused a decrease in the V value, possibly due to configurational alterations of the binary or ternary coenzyme complexes of the enzymes resulting in decreased cofactor activity of NAD+, but the decrease in the activity was somewhat compensated by the decrease in the values of Ka, Kb, and Kia also caused by the modification; (b) the incorporation of the NAD+ derivative into the side chain of polyacrylamide caused decrease in the affinity for the enzyme and further decrease in the V value, and thus decreased the cofactor activity to below that of the monomeric NAD+ derivative.