Influence of Tris(Hydroxymethyl)Aminomethane on Kinetic Mechanism of Yeast Alcohol Dehydrogenase

Abstract

Acetaldehyde, propionaldehyde, glyceraldehyde-3-P and 4-dimethylaminocinnamaldehyde form Schiff bases in Tris.HC1 buffers; the rates of formation and dissociation of Schiff bases, and equilibrium constants for their formation are very similar for the first three aldehydes. The steady-state kinetic constants for the yeast alcohol dehydrogenase-catalyzed reaction, propan-l-ol + NAD⁺ = propionaldehyde + NADH + H⁺, have been determined in several Tris.HCl buffers of increasing concentration at pH 8.1. In the forward direction, oxidation of alcohol, most kinetic constants are increased by increasing concentrations of Tris. In the reverse direction, reduction of aldehyde, substrate, NADH, Tris and Schiff base were equilibrated before enzyme reaction was started. It was found that Schiff base, rather than Tris, binds to free enzyme competitively with respect to NADH. Tris and Schiff base do not influence the binding of aldehyde to enzyme in any way.

Key Words:

• Yeast alcohol dehydrogenase
• Schiff base
• YADH-yeast alcohol dehydrogenase
• DACA-4-dimethyl-amino-cinnamaldehyde
• AA-acetamide