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, 2717-27211. 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 K(m)(app)NADP) 62-fold and increases k(cat)/K(m)(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.