Synergism Between Coenzyme and Carboxylate Binding to Liver Alcohol Dehydrogenase

Abstract

1. The interaction of decanoate and benzoate with the substrate-binding site in liver alcohol dehydrogenase has been characterized by fluorimetric equilibrium binding studies, using auramine 0 as a reporter ligand.2. The affinity of the enzyme for the carboxylates examined decreases about 50-fold on complex formation with NADH. The coenzyme-competitive inhibitors ADP-ribose and Pt(CN)a-have a similar destabilizing effect on decanoate binding, indicating that the effect of NADH derives from its negatively charged pyrophosphate group.3. Carboxylate binding to free enzyme requires the protonated form of an ionizing enzymic group with pKa 9.2, while there is no corresponding effect of pH on binary complex formation with auramine 0. Carboxylate binding to the enzyme . NADH complex exhibits no significant dependence on pH over the pH range 7 -10.4. These results, combined with previously reported data for decanoate binding to the enzyme . NAD' complex, provide clear evidence that carboxylates (in contrast to auramine 0) are bound at the active-site zinc ion of the enzyme in a process regulated by the ionization state of zinc-bound water. The synergistic effect of NADH and NAD' on carboxylate binding can be qualitatively and quantitatively explained in terms of electrostatic interactions analogous to those proposed to account for the effect of coenzymes on the pKa of zinc-bound water.Coenzyme binding to liver alcohol dehydrogenase between pH 6 and 10 is strongly affected by two protonation equilibria involving, respectively, free enzyme (pKa 9.2) and the binary enzyme . NAD+ complex (pKa 7.6) [1,2]. It appears well established that the pKa-7.6-dependence of NAD' binding derives from ionization of the water molecule known to be bound at the active-site zinc ion of the enzyme subunit [I]. Recent investigations of the effect of pH on complex formation with anions and zinc-chelating reagents [3,4] led to the conclusion that ionization of zinc-bound water is likely to account also for the pKa-9.2-dependence of NAD+ and NADH association to free enzyme. 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. The corresponding destabilizing interaction in the enzyme . NAD+ complex was envisaged to be counteracted by the stabilizing effect of the positively charged nicotinamide ring of the coenzyme. This was assumed to result in a net perturbation of the pK, of zinc-bound water from 9.2 to 7.6 on the binding of NAD'.There are important mechanistic implications of the proposal that differences in magnitude of the pKa values regulating coenzyme binding to liver alcohol dehydrogenase reflect electrostatic interactions between coenzyme and ...

Electrostatic effects of bound NADH and NAD+ on ionizing groups in liver alcohol dehydrogenase

“…The kinetic evidence indicating that the corresponding group exhibits a pK, of 9.2 in the absence of bound coenzyme [4,17,291 is strongly supported by data in Tables 1 and 4, which establish that NADH binding must be assumed to cause a most significant decrease in acidity of zinc-bound water due to the electrostatic effect of the negatively charged pyrophosphate group of the coenzyme. The theoretically estimated magnitude (1.5) of this pK, shift is in satisfactory agreement with the pKa shift of 2.0 deduced from pH-dependence data for NADH binding [5], as well as with the pK shifts of 1.7 reported for the NADH-induced destabilization of cyanide [26] and decanoate [27] binding to catalytic zinc.…”

Hydrophobic anion activation of human liver .chi..chi. alcohol dehydrogenase

“…Modeling of the primary structure of xx-ADH to that of the horse enzyme indicates that there are extensive substitutions making the hydrophobic barrel of xx-ADH more hydrophilic (Figure 9). The catalytic zinc atom is not the likely binding locus since such a mode of binding will result in inhibition as has been observed with human class I and the horse EE isozymes (Andersson et al, 1981). The inhibition of xx-ADH observed with some large substrates at high activator concentrations suggests that direct binding to zinc is possible, however.…”

Alternative pathways and reactions of benzyl alcohol and benzaldehyde with horse liver alcohol dehydrogenase

“…Benzoate is not reduced by NADH, but it binds to the enzyme-NADH complex with a K\ of 150 (±30) mM (Andersson et al, 1981). It is also a competitive inhibitor against ethanol or benzyl alcohol, with a dissociation constant of 14 (±3) mM with 1 mM NAD+ in 46 mM sodium phosphate buffer and 0.25 mM EDTA (pH 7) at 30 °C.…”

“…The oxidation of benzaldehyde to benzoic acid is irreversible, so that a very small rate constant was fixed for the simulation of the dismutation. /The rate constants were chosen arbitrarily such that the ratio is equivalent to the Ki of 150 ± 30 mM reported in the literature(Andersson et al, 1981). *The rate constants were chosen arbitrarily such that the ratio is equivalent to the Ki determined experimentally in this work, 14 mM.…”