INVOLVEMENT OF THE C-TERMINAL TAIL IN THE ACTIVITY OF DROSOPHILA ALCOHOL-DEHYDROGENASE - EVALUATION OF TRUNCATED PROTEINS CONSTRUCTED BY SITE-DIRECTED MUTAGENESIS

Drosophila alcohol dehydrogenase belongs to the heterogeneous family o f short-chain dehydrogenases/reductases, which does not include the we ll characterized mammalian alcohol dehydrogenases. Although it is clea r that the main biological role of this enzyme is in alcohol oxidation , in the absence of the three-dimensional conformation only partial in formation on the protein regions involved in the active site, and the coenzyme and substrate interacting cavities is available. Two segments have already been identified, a coenzyme-binding segment at the N-ter minus, and the reactive Tyr152 and Lys156 residues. Limited proteolyti c assays had suggested the involvement of the 13 C-terminal amino acid s in the function of the enzyme. By site-directed mutagenesis, we have constructed eight different truncated mutant enzymes and expressed th em in Escherichia coli. The purified mutant enzymes have been recovere d and characterized using monoclonal antibodies. Kinetic analysis and stability assays have been performed, and clearly demonstrate the cont ribution of the last 13 amino acids to the activity. We hypothesize th at the C-terminal tail constitutes an essential region for maintaining the hydrophobicity of the catalytic pocket needed for binding of the substrate.