Aldoximes: Active‐Site Probes of Alcohol Dehydrogenases

The oximes of aliphatic aldehydes inhibit horse liver and yeast alcohol dehydrogenase. The pattern of inhibition of these enzymes by the oximes reflects their substrate specificity. For example, acetaldoxime is a more effective inhibitor of the yeast enzyme than butyraldoxime (K1 0.06 mM and 2 mM respectively). However, for the liver enzyme, the K1 for butyraldoxime is 6.6 nM while that for acetaldoxime is 0.68 μM. The inhibition constants of hexaldoxime, octaldoxime and decaldoxime for the liver enzyme are 2 nM, 1 nM and 0.7 nM, respectively. The inhibition of the liver enzyme by the aliphatic oxime is attributable to the anti isomer which forms spectroscopically identifiable ternary complexes with the enzyme and NAD+. All complexes exhibit a transition with a maximal absorption from 290–305 nm and an absorption coefficient of approximately 7 mM−1 cm−1 which most likely results from the addition of the hydroxyl group of the oxime to the nicotinamide moiety of the coenzyme. An analogous complex is not formed with yeast alcohol dehydrogenase. Since the dissociation constants determined from steady‐state inhibition data and the kinetics of formation and dissociation of the spectroscopically identifiable complex are identical, the enzyme‐NAD+‐oxime complex must be exclusively responsible for the observed inhibition, even though ternary complexes with NADH also form. The ternary complex formed with the oxime of p‐dimethylaminocinnamaldehyde has the characteristic absorption at 290–305 nm, indicative of oxygen addition to the nicotinamide moiety, and in addition exhibits a spectral shift in its long‐wavelength transition from 330 nm to 425 nm, suggestive of inner‐sphere coordination of the zinc ion by the nitrogen of the oxime. Anti‐oximes are structurally homologous to 4‐alkyl pyrazoles which are also very effective inhibitors of horse liver alcohol dehydrogenase. The relative insensitivity of the yeast enzyme to pyrazole and oximes suggests that the catalytic function of the zinc ion in these two enzymes cannot be assumed to be identical.