Flow kinetics of yeast alcohol dehydrogenase attached to nylon tubing

Yeast alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was attached covalently to the inner surface of nylon tubing, and the immobilized enzyme retained its activity over a period of months. A study was made of the flow kinetics for the reaction between ethanol and NAD. With the ethanol held at saturating concentrations there was partial diffusion control, the extent decreasing with increasing flow rate and increasing NAD concentration. With the NAD at saturating concentrations there was no appreciable diffusion control. The apparent Michaelis constants varied with flow rate vf, being linear in vf-1/3, and extrapolation to infinite flow rate (vf-1/3 = 0) gave the intrinsic Michaelis constants. The inhibition by products was also studied. The results for both NADH and acetaldehyde showed mixed competitive and non-competitive inhibition, with a preponderance of the former. Acetaldehyde is the stronger inhibitor, and this is consistent with the lack of dissusion control with variable ethanol. Inhibition by acetaldehyde is not affected by flow rate, but inhibition by NADH is affected, presumably because of the greater degree of diffusion control with variable NAD.