Binding of Inhibiting Adducts of Ketones and NAD+to Alcohol Dehydrogenase fromDrosophila melanogaster

Drosophilaalcohol dehydrogenase (DADH) can be converted with NAD+and ketone into more negatively charged isoforms. Completely modified ADH isoforms are inactive, but activity is regained after native polyacrylamide gel electrophoresis depending on the ketone used. When unmodified ADH is incubated with NAD+and acetylacetone, more negatively charged bands appear. Modified ADH isoforms bind to a Mono-Q column, while unmodified ADH and NAD+do not. A covalently bound adduct is also formed between NAD+and ketone in the absence of enzyme. These adducts can be purified by ion-exchange FPLC and have been characterized by mass spectrometry and UV spectroscopy and also form inactive isoforms after incubation with unmodified enzyme. The effect of temperature increase on unmodified and modifiedDrosophila melanogastervariants was determined by circular dichroism experiments. Unfolding temperatures (Tm) of the modified ADH isoforms were 5 to 20° higher than those of the unmodified ones, indicating that the enzyme molecule becomes more compact upon adduct binding.