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Interaction of the cofactor NAD with its radical anion and its role in the activation of yeast alcohol dehydrogenase 1 (YAD1).
The NAD(+) cofactor is required for the activation of alcohol dehydrogenases (ADHs) by means of a ping-pong mechanism. Previous studies established that the mechanism starts at the alcohol substrate and that the presence of NAD(+) was required for the electron transfer from NAD(H) via the NAD/NADH redox couple to the flavin of the active site. The kinetics of the subsequent oxidation of the flavin redox couple and the mechanism by which NAD(+) radical anion formation triggers ADH activation are still debated. In this work, we quantified the amount of NAD(+) radical anion in equilibrium with NAD(+) by continuous-flow spectrophotometry and established that the equilibrium is rapidly reached. The formation of the NAD(+) radical anion is the rate-limiting step in the activation of YAD1 and requires the presence of NAD(+) in the reaction mixture. The pH-dependence of the formation of the NAD(+) radical anion, expressed as the k(rad) observed in
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