Alcohol dehydrogenase from the fruitfly Drosophila melanogaster Inhibition studies of the alleloenzymes Adh S and Adh UF

Abstract

Different metal binding inhibitors of horse liver alcohol dehydrogenase, similarly affect the Drosophila melaogaster Adh s and Adh UF alleloenzymes. However, binding is generally weaker and the experiments show that the alleloenzymes although not zinc metalloenzymes, behave to the metal binding reagents very munch as if the were. The metal-directed, affinity-labelling, imidazole derivative BrImPpOH reversibly inhibits, but does not inactivate the alleloenzymes. This confirms there is no active site metal atom with cysteine as a metal ligand, as found in zinc alcohol dehydrogenases. Pyrazole is a strong ethanol-competitive inhibitor of the Adhs s and Adh UF alleloenzymes. Formation of the ternary enzyme-NAD-pyrazole complex gives an absorption increase between 295–330 nm. This enables an active site titration to be performed and the determination of ϵ(305 nm) of 15.8·10 3 M −1·cm −1. Inhibition experiments with imidazole confirm that with secondary alcohols such as propan-2-ol, a Theorell-Chance mechanism predominates, but with ethanol and primary alcohols, interconversion of file ternary complexes is rate limiting. Salicylate is a coenzyme competitive inhibitor and K EI suggests that the coenzyme adenosine binding region is similar in Drosophila and horse liver alcohol dehydrogenase. Drosophila alcohol dehydrogenase is found not to form a ternary complex with NADH and isobutyramide. In this and other properties it is like carboxymethyl liver alcohol dehydrogenase. Both Drosophila and carboxymethyl alcohol dehydrogenase bind coenzyme in a similar manner to native horse liver alcohol dehydrogenase, but substrate binding differs between each. Inhibition by Cibacrone blue, indicates that amino acid 192 which is lysine in Adh s and threonine in Adh UF, is located in the coenzyme-binding region. Proteolytic activity present in preparations of alcohol dehydrogenase from D. melanogaster, is considered due to a metalloprotease, for which BrImPpOH is a potent inactivator.