Evidence that Ionization of Zinc‐Bound Water Regulates the Anion‐Binding Capacity of the Coenzyme‐Binding Site in Liver Alcohol Dehydrogenase

Abstract

1. The mechanistic and structural origin of the pKa 9.2 dependence of coenzyme association and anion binding to liver alcohol dehydrogenase has been investigated by titrimetric and spectrophotometric binding studies involving ligands (imidazole, 1,lO-phenanthroline and 2,2'-bipyridine) which combine to the catalytic zinc ion of the enzyme subunit with displacement of zinc-bound water.2. Imidazole abolishes the pKa9.2 dependence of NADH binding to the enzyme. The pH dependence of ADP-ribose and Pt(CN)$-binding is similarly abolished by imidazole, as well as by 1,lO-phenanthroline and 2,2'-bipyridine. It is concluded from these results that the pKa 9.2 dependence of coenzyme and anion binding most likely derives from ionization of zinc-bound water.3. Evidence is presented showing that the protonation state of the pK, 9.2 group also regulates ligand binding to the catalytic zinc ion, which lends strong support to the conclusion that this ionizing group can be identified as zinc-bound water. The pK, 9.2 dependence of bipyridine binding is abolished by ADP-ribose and Pt(CN)?-, indicating that complex formation at the anion-binding subsite of the coenzyme-binding site perturbs the pKa of zinc-bound water to a value above 10.4. The cooperative interrelationship between ionization of zinc-bound water and complex formation at the anion-binding subsite is proposed to be attributable to a competition between the zincbound hydroxyl ion and external anionic ligands for salt bridge formation with the guanidinium group of Arg-47. This explains why ionization of zinc-bound water affects the anion-binding capacity of the enzyme and why complex formation at the anion-binding subsite affects the pKa of zinc-bound water.5. The kinetics of complex formation with bipyridine are consistent with a two-step binding mechanism in which a pKa 9.2 dependent rapid pre-equilibration between enzyme and ligand is followed by a pH independent rate-limiting formation of the chromophoric binary complex. Desorption of zinc-bound water, therefore, is likely to take place in the primary association step rather than in the subsequent rate-limiting step. This renders the possibility less likely that productive ternary complexes formed during catalysis may contain a zinc-bound penta-coordinate water molecule.In a recent report from our laboratory it was shown that the proton dissociation mechanism in Scheme 1 accounts for the main effects of pH on coenzyme binding to liver alcohol dehydrogenase over the pH range 6-10 [l]. The ionization step involving free enzyme (pKa 9.2) was found not only to control the rates of NADH and NAD' association to the enzyme, but also to regulate the binding of coenzyme-competitive anions such as Pt(CN)i-and the coenzyme fragments ADP-ribose and AMP. Since there is no Enzyme. Liver alcohol dehydrogenase or alcohol : NAD' oxidoreductase (EC 1.1.1.1). similar effect of pH on binary-complex formation with adenosine [2], it was concluded that the pKa 9.2 dependence of coenzyme association reflects ionization of a functional gro...

Electrostatic field effects of coenzymes on ligand binding to catalytic zinc in liver alcohol dehydrogenase

“…A fit of Eqns (1) and (2) to data in Fig. 3 gave Kapp = 23 ( & 3) pM, however, which corresponds to a cooperativity factor CI = 48 in Eqn (3). Similar analysis of data obtained with 1.2mM CN-gave Kapp = 17pM and o!…”

“…The kinetics of bipyridine binding to the enzyme were examined as described by Anderson et al [3]. Complex formation between enzyme (1 -30pM) and NADi (0.5 -200pM) was monitored photometrically at 275 nm by stopped-flow techniques.…”

“…Since a zinc-bound cyanide ion is unlikely to interfere sterically with coenzyme binding [l], the observed cooperativity effects can be assumed to derive almost exclusively from electrostatic interactions. According to previously advanced ideas [3], charge interactions of zinc-bound CN-with the coenzyme pyrophosphate group (probably mediated by the side chain of Arg-47) are likely to provide a major contribution to the observed destabilization of NADH binding. Stabilization of the binding of NAD' is certainly achieved through charge interaction of CN-with the coenzyme nicotinamide ring, which is bound in close proximity to catalytic zinc [l].…”

“…Bipyridine binding to the enzyme.CN-complex was found to be rate-limited by an apparent first-order process with a rate constant tending towards 25 s-with increasing bipyridine concentrations. According to the kinetic theory for displacement reactions and reported data for bipyridine binding [3], the latter value can be taken to provide a comparatively precise estimate of the rate constant for CN-dissociation from the enzyme. This estimate corresponds (Table 1) CN-Complex formation between enzyme and C N -was found not to be associated with any detectable absorption changes in the 300 -800-nm region.…”

“…Such discussions have focused on the identity and mechanistic significance of the ionizing groups which control coenzyme and substrate binding to the enzyme. We have attributed the observed pH dependence of coenzyme binding to electrostatic interactions reflecting ionization of the water molecule which is bound at the catalytic zinc ion of the enzyme subunit [3]. Charged groups on NADH and NAD' were assumed to interact strongly enough with a zinc-bound hydroxyl ion to account for the pK, values assigned to zincbound water in different enzyme species [4].…”