Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase

Abstract

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 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.