Open AccessRole of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase
Author(s) -
CHEN Zhuo,
LEE William R.,
CHANG Simon H.
Publication year - 1991
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1991.tb16371.x
Subject(s) - nad+ kinase , cofactor , alcohol dehydrogenase , dehydrogenase , enzyme , biochemistry , stereochemistry , biology , oxidoreductase , chemistry
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.