Rolf Hovik

The alcohol dehydrogenase alleloenzymes AdhS and AdhF from the fruitfly Drosophila melanogaster: an enzymatic rate assay to determine the active-site concentration.

A rapid and reproducible enzymatic rate assay for the quantitative determination of the concentration of active sites is presented for the alleloenzymes AdhS and AdhF from Drosophila melanogaster. Using this procedure the turnover numbers as catalytic-center activities were found to be 12.2 sec−1 for AdhF and 3.4 sec−1 for AdhS with secondary alcohols. This showed a slower dissociation of the coenzyme from the binary enzyme-NADH complex with AdhS and hence a stronger binding of NADH to this alleloenzyme. With ethanol, the catalytic-center activity was 1.4 sec−1 for AdhS and 2.8 sec−1 for AdhF, and hence the single amino acid mutation distinguishing the two alleloenzymes also affected hydride transfer.

Drosophila melanogaster alcohol dehydrogenase: Substrate sterospecificity of the AdhF alleloenzyme

Abstract The substrate specificity and the stereospecificity of the AdhF alleloenzyme from Drosophila melanogaster have been investigated. These properties like the topology of the alcohol binding region and the mechanism of oxidation of primary and secondary alcohols are found to be similar to that of the AdhS and AdhUF alleloenzymes. AdhF like AdhUF, binds the coenzyme-competitive inhibitor Cibacron blue much more weakly than does the AdhS alleloenzyme. This shows that amino acid residue 192 affects coenzyme binding. It also accounts for the observed differences between the activities of these alleloenzymes with respect to their maximum velocity ( V m ) with secondary alcohols as substrates.