Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase.
Drosophila alcohol dehydrogenase (ADH), an NAD(+)-dependent dehydrogenase, shares little sequence similarity with horse liver ADH. However, these two enzymes do have substantial similarity in their secondary structure at the NAD(+)-binding domain [Benyajati, C., Place, A. P., Powers, D. A. & Sofer, W. (1981) Proc. Natl Acad. Sci. USA 78, 2717-2721]. Asp38, a conserved residue between Drosophila and horse liver ADH, appears to interact with the hydroxyl groups of the ribose moiety in the AMP portion of NAD+. A secondary-structure comparison between the nucleotide-binding domain of NAD(+)-dependent enzymes and that of NADP(+)-dependent enzymes also suggests that Asp38 could play an important role in cofactor specificity. Mutating Asp38 of Drosophila ADH into Asn38 decreases Km(app)NADP 62-fold and increases kcat/Km(app)NADP 590-fold at pH 9.8, when compared with wild-type ADH. These results suggest that Asp38 is in the NAD(+)-binding domain and its substituent, Asn38, allows Drosophila ADH to use both NAD+ and NADP+ as its cofactor. The observations from the experiments of thermal denaturation and kinetic measurement with pH also confirm that the repulsion between the negative charges of Asp38 and 2'-phosphate of NADP+ is the major energy barrier for NADP+ to serve as a cofactor for Drosophila ADH.
Chen
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Department of Biochemistry, Louisiana State University, Baton Rouge 70803.
Lee
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Chang
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D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D004330:Drosophila / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006736:Horses; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011489:Protein Denaturation; D012689:Sequence Homology, Nucleic Acid
D009243:NAD; D001224:Aspartic Acid; D009249:NADP; D000426:Alcohol Dehydrogenase