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

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Abstract
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.