Reaction of 4-trans-(N,N-dimethylamino)cinnamaldehyde with the liver alcohol dehydrogenase-oxidized nicotinamide adenine dinucleotide complex

Evidence that horse liver alcohol dehydrogenase forms a ternary complex with 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) and oxidized nicotinamide adenine dinucleotide (NAD+) is presented. Formation of the complex is characterized by a 97-nm red shift of the free chromophore to 495 nm (epsilon 495 approximately 6.0 X 10(4) M-1 cm-1). This shift is larger than the 66-nm red shift of the E(NADH,-DACA) complex (lambda max = 464 nm) previously reported by Dunn and Hutchinson [Dunn, M.F., & Hutchison, J.S. (1973) Biochemistry 12, 4882-4892]. The large red shift of the E(NAD+,DACA) complex is due to the combined effects of coordination of the carbonyl oxygen of DACA to the active-site zinc ion and to the close proximity of the positively charged nicotinamide ring of NAD+. The stability of this complex is pH dependent and depends on a single apparent ionization with pKa = 7.6 +/- 0.3. The pH-independent dissociation constant for binding of DACA to E(NAD+) is 23 +/- 6 microM. The stoichiometry of DACA binding to the E(NAD+) complex is shown to be one per active site (two per enzyme molecule). Liver alcohol dehydrogenase is also shown to catalyze the NAD+-mediated oxidation of DACA to the corresponding carboxylic acid with a very slow turnover rate. The possibility that the observed E(NAD+,DACA) complex is an intermediate in the enzyme-catalyzed oxidation of DACA is discussed.