Untersuchungen zur Reaktion der Alkoholdehydrogenase mit Tritium -markierten Substraten

Abstract

The temperature dependence of the kinetic isotope effect of NADH-T in the acetaldehyde reduction by yeast alcohol dehydrogenase showed a discontinuity which can be explained by a change of the rate controlling step. The magnitude of the isotope effect is largely dependent on the nature of the unlabelled aldehyde and increases in the order acetaldehyde, propionaldehyde, butyraldehyde. This is a direct indication that the second substrate influences the nature of the H-transfer from NADH. During substrate binding the aldehyde causes an effect on the transferable H of NADH. This effect is less pronounced for the less effective substrates propionaldehyde and butyraldehyde. Comparing the homologue aldehydes the small size of the isotope effect gives an indication that acetaldehyde is the natural substrate of yeast alcohol dehydrogenase. The purification of NADH-T on DEAE-Cellulose is connected with isotope fractionation which amounts to 0.4 — 1.1% of retention volume.