Effect of NADH on the pKa of Zinc‐Bound Water in Liver Alcohol Dehydrogenase

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Abstract
Equilibrium constants for coenzyme binding to [horse] liver alcohol dehydrogenase was determined over the pH range 10-12 by pH-jump stop-flow techniques. The binding of NADH or NAD+ requires the protonated form of ionizing group (distinct from Zn-bound water), with a pKa of 10.4. Complex formation with NADH exhibits an additional dependence on the protonation state of an ionizing group, with a pKa of 11.2. The binding of trans-N,N-dimethylaminocinnamaldehyde to the enzyme.cntdot.NADH complex is prevented by ionization of the latter group. The pKa-11.2-dependence of NADH binding most likely derives from ionization of the water molecule bound at the catalytic Zn+ of the enzyme subunit. The pKa value of 11.2 was thus assigned to Zn-bound water in the enzyme. NADH complex appears to be typical for an aquo ligand in the inner-sphere ligand field, provided by the Zn-binding amino acid residues in liver alcohol dehydrogenase. This means that the pKa of metal-bound water in Zn-containing enzymes can be assumed to correlate primarily with the number of negatively-charged protein ligands coordinated by the active-site Zn+.

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