BINDING OF FORMAMIDES TO LIVER ALCOHOL-DEHYDROGENASE

Amides are analogs of aldehydes and potent inhibitors of liver alcohol dehydrogenases. They can be used for structural studies and for inhib iting the metabolism of alcohols that form toxic products. We studied N-alkyl amides that bind to the enzyme-NADH complex and act as uncompe titive inhibitors against varied concentrations of ethanol (millimolar K-ii values, at pH 8 and 25 degrees C): N-propylacetamide (16), delta -valerolactam (1.6), N-formylpiperidine (0.14), N-isobutylformamide (0 .028), N-(cyclohexylmethyl)formamide (0.011), and N-cyclohexylformamid e (0.0087). The lower affinity of delta-valerolactam and N-propylaceta mide can be explained by steric hindrance with Phe93 of the enzyme. Re placing Phe93 with Ala in the S48T/F93A mutated enzyme, which resemble s the natural alpha-isoenzyme of primates, improved binding of delta-v alerolactam by 210-fold. The structures of horse liver enzyme complexe d with NADH and N-cyclohexylformamide or N-formylpiperidine were deter mined by X-ray crystallography at 2.5 Angstrom resolution. In both com plexes, the carbonyl oxygens of the inhibitors bind to the catalytic z inc and form a hydrogen bond to the hydroxyl group of Ser48 of the enz yme. The six-membered rings bind in overlapping, but rotated, position s that optimize hydrophobic interactions. The binding modes of the unr eactive formamides appear to resemble the Michaelis complexes of the a nalogous substrates, with the re face of the carbonyl carbon suitably positioned to accept a hydrogen from NADH.