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

Abstract

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' requires the protonated form of an ionizing group (distinct from zinc-bound water) with a pK, of 10.4. Complex formation with NADH exhibits an additional dependence on the protonation state of an ionizing group with a pK, of 11.2. The binding of trans-N,N-dimethylaminocinnamaldehyde to the enzyme . NADH complex is prevented by ionization of the latter group.It is concluded from these results that the pKd-ll.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 pK, 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 pK, of metal-bound water in zinccontaining enzymes can be assumed to correlate primarily with the number of negatively charged protein ligands coordinated by the active-site zinc ion.Substrate and coenzyme binding to liver alcohol dehydrogenase appears to be drastically affected by ionization of the water molecule which is bound at the catalytic zinc ion of the enzyme subunit [l -61. In a recent investigation of the pH dependence of ligand binding at the catalytic zinc ion [7] we concluded that zinc-bound water exhibits a p y of 9.2 in free enzyme, decreasing to 7.6 in the enzyme . NAD+ complex and increasing to a value above 10 in the enzyme . NADH complex. We have now attempted to obtain more detailed information about the effect of complex formation with NADH on the pK, of zinc-bound water by examination of the effect of pH on NADH binding at pH values above 10. The results described in the present paper show that complex formation between enzyme and NADH is regulated by a proton dissociation equilibrium with a pK, of 11.2, while there is no corresponding effect of pH on NAD' binding over the examined pH range (10-12). We propose that the observed pK,-lI .2-dependence of NADH binding derives from ionization of zinc-bound water in the binary complex formed. Equilibrium data for aldehyde binding to the enzyme . NADH complex are reported which lend strong support to the latter proposal.

Structures of Shikimate Dehydrogenase AroE and Its Paralog YdiB

“…6A). Another possibility is the involvement of Thr 61 , the 2Ј-hydroxyl of the cofactor, and His 13 in a proton relay analogous to that found in alcohol dehydrogenase (43). The pH dependence of AroE (maximum at pH 7.3) is consistent with a histidine being involved in the mechanism.…”

Synergism Between Coenzyme and Carboxylate Binding to Liver Alcohol Dehydrogenase

“…NADH complex. This pH dependence pattern has been proposed to reflect that complex formation involving ligand binding to the activesite zinc ion is prevented by ionization of the zinc-bound water molecule [I, 6,181. The present results are obviously consistent with that idea.…”

“…This would imply that the pKa of zinc-bound water is perturbed from 9.2 to a value above 10 on the binding of NADH [2,5], and evidence has been presented indicating that zinc-bound water actually exhibits a pKa of 11.2 in the enzyme . NADH complex [6]. Andersson et al [4] attributed this shift in pKa to destabilization of the zinc-bound hydroxyl ion through (indirect) electrostatic interaction with the negatively charged pyrophosphate group of NADH.…”

On the Molecular Mechanism of Liver Alcohol Dehydrogenase: Monte Carlo Simulations and X-Ray Studies of Water in the Substrate Channel

“…NAD+ binding is associated with a pKa shift of the zinc-bound water from 9.2 down to 7.6 [18]. This pKa in presence ofNADH is 11.2 [18].…”

“…NAD+ binding is associated with a pKa shift of the zinc-bound water from 9.2 down to 7.6 [18]. This pKa in presence ofNADH is 11.2 [18]. The pH dependence of the catalytic activity can be traced back to a protein-coenzyme modulated self-dissociation of water: (4) or by using W5 in the active site one can form the channel Zn(0H2)...H20...H20 -> Zn(0H-)...0H2...H30+ (5) The theoretically calculated LADH elec tric field shows a gradient favoring a proton release from the active site towards the sur face.…”