Description:
<jats:p>Equilibrium constants for coenzyme binding to liver alcohol dehydrogenase have been determined over the pH range 10–12 by pH‐jump stop‐flow techniques. The binding of NADH or NAD<jats:sup>+</jats:sup> requires the protonated form of an ionizing group (distinct from zinc‐bound water) with a p<jats:italic>K</jats:italic><jats:sub>a</jats:sub> of 10.4. Complex formation with NADH exhibits an additional dependence on the protonation state of an ionizing group with a p<jats:italic>K</jats:italic><jats:sub>a</jats:sub> of 11.2. The binding of <jats:italic>trans‐N,N</jats:italic>‐dimethylaminocinnamaldehyde to the enzyme · NADH complex is prevented by ionization of the latter group.</jats:p><jats:p>It is concluded from these results that the p<jats:italic>k</jats:italic><jats:sub>a</jats:sub>‐11.2‐dependence of NADH binding most likely derives from ionization of the water molecule bound at the catalytic zinc ion of the enzyme subunit. The p<jats:italic>K</jats:italic><jats:sub>a</jats:sub>, value of 11.2 thus assigned to zinc‐bound water in the enzyme · NADH complex appears to be typical for an aquo ligand in the inner‐sphere ligand field provided by the zinc‐binding amino acid residues in liver alcohol dehydrogenase. This means that the p<jats:italic>K</jats:italic><jats:sub>a</jats:sub> of metal‐bound water in zinc‐containing enzymes can be assumed to correlate primarily with the number of negatively charged protein ligands coordinated by the active‐site zinc ion.</jats:p>