Modeling Studies of Conformational Changes in the Substrate-Binding Loop in Drosophila Alcohol Dehydrogenase

Three-dimensional structures of sevenshort-chain dehydrogenases/reductases show that theseenzymes share common structural features. Sequencealignment studies of Drosophila alcoholdehydrogenase (DADH), with an unknown 3D-structure, and fourshort-chain dehydrogenases/reductases with known X-raystructures suggest that DADH shares the same structuralfeatures. However, the substrate binding regions, which are located in the C-terminal region of theseenzymes, share little sequence homology, because of thewide variety of substrates used. X-ray structures ofshort-chain dehydrogenases/reductases indicate that conformational changes occur in a loop, inthe C-terminal region, upon substrate binding. Thissubstrate-binding loop is located between a strand anda helix and may contain one or two small helices. Secondary structure predictions and modelingstudies of this substrate-binding loop in DADH predictthat the two helices may also be present in this enzyme.The naturally occurring variants of Drosophila melanogaster alleloenzymes ADH-S and ADH-Fdiffer in a replacement of threonine by lysine atposition 192, which is located at a central position inthe substrate-binding loop. The positive charge oflysine may move significantly on substrate binding,resulting in a direct charge interaction withNAD⁺ in the enzyme-substrate complex,explaining a very strong influence of pH on the bindingof ADH-S for the NAD⁺ analogue Cibacron Blue. Thisindicates that the ADH S/F polymorphism has a directinfluence on the catalytic properties of the enzyme.