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 of short-chain dehydrogenases/reductases, which does not include the well characterized mammalian alcohol dehydrogenases. Although it is clear that the main biological role of this enzyme is in alcohol oxidation, in the absence of the three-dimensional conformation only partial information 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-terminus, and the reactive Tyr152 and Lys156 residues. Limited proteolytic assays had suggested the involvement of the 13 C-terminal amino acids in the function of the enzyme. By site-directed mutagenesis, we have constructed eight different truncated mutant enzymes and expressed them in Escherichia coli. The purified mutant enzymes have been recovered and characterized using monoclonal antibodies. Kinetic analysis and stability assays have been performed, and clearly demonstrate the contribution of the last 13 amino acids to the activity. We hypothesize that the C-terminal tail constitutes an essential region for maintaining the hydrophobicity of the catalytic pocket needed for binding of the substrate.
Albalat
R
R
Department of Genetics, Faculty of Biology, University of Barcelona, Spain.
Valls
M
M
Fibla
J
J
Atrian
S
S
Gonzàlez-Duarte
R
R
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004330:Drosophila / Q000201:enzymology*; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase