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

A method for the synthesis of NAD + ‐ N 6 ‐[ 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 ( K a ) and for substrate ( K b ), and the dissociation constant of the enzyme‐coenzyme complex ( K ia ). In the reaction system with the monomeric NAD + derivative and yeast alcohol dehydrogenase, the K ia value was similar to that for NAD + and the values of V, K a , and K b 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, K a , K b , and K ia 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, K a , K b and K ia 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 K a , K b , and K ia 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.