Yeast alcohol dehydrogenase IV. Binding of reduced coenzyme and its fragments.

• 1.1. Commercial preparations of the enzyme are homogeneous on an ion-exchange column chromatography they are heterogeneous with respect to their zinc content, total -SH content, state of oxidation and the reactivity of their -SH groups.
• 2.2. Heterogeneous properties of commercial preparations have not influenced their coenzyme binding capacity. All commercial preparations tested in this, and our earlier work (Leskovac &; Pavkov-Peričin, 1975; Leskovac et al., 1976), bind approx. 2 molecules of NADH/tetrameric enzyme molecule (mol. wt. 144,000), in a non-cooperative fashion. We have arrived at this binding capacity by 3 different methods: fluorescent titration, gel-filtration and the rate enhancement of the Ellman reaction in the presence of coenzyme.
• 3.3. pH-profile of the coenzyme dissociation constant indicates that 2 charged groups on the enzyme, with pK. 7.4 and 9.4, control the enzyme-coenzyme complex formation.
• 4.4. Commercial preparations bind weakly coenzyme fragments, adenosyl pyrophosphate ribose or ADP, with a stoichiometry of 4–5 fragment molecules/tetrameric molecule of enzyme.
• 5.5. After the binding, the coenzyme or its fragments induce a conformational change in the enzyme; the magnitude of this conformational change decreases in the following order: NADH + acetamide > NADH > adenosyl pyrophosphate ribose > ADP.