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

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Abstract
The Z isomer of 4-trans-(N,N-dimethylamino)cinnamaldoxime, (Z)-DMOX (.lambda.maxH2O 354 nm), forms a ternary complex with NAD+ and equine liver alcohol dehydrogenase. The 3-acetyl (3-acetyl-PdAD+), 3-thiocarboxamide (3-thio-NAD+), 3-iodo (io3PdAD+) and nicotinamide mononucleotide (NMN+) analogs of NAD+ also form ternary complexes with enzyme and (Z)-DMOX. These complexes are characterized by large red-shifts in the UV-visible spectrum of bound (Z)-DMOX (.lambda.max 428 nm for the NAD+ complex) and new spectral bands in the 280-340-nm region associated with the pyridine moieties of NAD+ and the NAD+ analogs. The ternary enzyme-NAD+-(Z)-DMOX complex is weakly fluorescent (.lambda.ex 430 nm; .lambda.emmax 505 nm) and strongly quenches the residual tryptophan fluorescence of the enzyme-NAD+ binary complex. (Z)-DMOX binds with high affinity to the enzyme-NAD+ complex (Kd .ltoreq. 4 .times. 10-9 M at pH 8.75 and 25.degree. C), and similarly high affinities were found for the 3-acetyl-PdAD+, 3-thio-NAD+ and io3PdAD+ complexes. Binding is much weaker to the enzyme-NMN+ complex. The active site specifically substituted Co(II), Ni(II), Cu(II) and Cd(II) enzyme derivatives and the enzyme species lacking any metal ion at the active site (apoenzyme) also form ternary complexes with (Z)-DMOX in which the DMOX UV-visible spectrum is red-shifted (ranging from 43 to 83.5 nm). The complexes formed with the Zn(II) and Co(II) enzymes are characterized by relatively high affinities for (Z)-DMOX and by spectra that are independent of pH over the range 6-10. The affinity of the apoenzyme-NAD+ complex for (Z)-DMOX is much lower, and the spectrum of the complex is pH dependent with .lambda.max = 430 nm at pH 7 and .lambda.max = 397 nm at pH 10. The rate of (Z)-DMOX dissociation from the apoenzyme complex was .apprx. 103-fold greater than the rates observed for the metal ion substituted enzymes. The 280-340-nm spectral bands appear to result from the dihydropyridine moieties of covalent adducts formed between (Z)-DMOX and NAD+ and the NAD+ analogs. The large red-shifts of the (Z)-DMOX spectrum result from the bonding of the oxime nitrogen to a strong electrophilic center (either the active site zinc ion or the nicotinamide ring of NAD+). Attempts to fit these structures into the 3-dimensional structure of the E(NADH,Me2SO) complex via model building indicate coordination of the oxime nitrogen to Zn and covalent bond formation between the oxime O and C-4 of the nicotinamide ring. The high affinities and slow rates of dissociation of the metal-substituted enzyme complexes are due in part to the coordination of (Z)-DMOX to the active site metal, an interaction that is not possible for the apoenzyme complex.