1. Kinetic and equilibrium data have been determined at different pH between 4 and 10 for binding of the inhibitor pyrazole to liver alcohol dehydrogenase and to the binary complexes formed between enzyme and NADH or NAD '. 2. Pyrazole binding to free enzyme requires the protonated form of an ionizing group with a pK, of 9.2, agreeing with the pKa value reported for the water molecule bound at the catalytic zinc ion of the enzyme subunit. The rate of association of the inhibitor to the enzyme . NAD' complex exhibits a similar pKa-7.6-dependence attributable to ionization of zinc-bound water in the latter binary complex. These observations lend support to the idea that pyrazole combines to the catalytic zinc ion on complex formation with the enzyme, zinc-bound water most likely being displaced by the inhibitor.3. The rate of dissociation of the inhibitor from the ternary enzyme . NAD' . pyrazole complex is proportional to the hydrogen ion concentration over the examined pH range (4-8). This effect of pH, which is proposed to reflect ionization of the enzyme-bound inhibitor with a pK, value below 4 (indirectly estimated to 2.4), accounts for the exceptional stability of the ternary complex at neutral and alkaline pH. It is concluded that pyrazole, by analogy to water and alcohol ligands, undergoes a drastic pK, perturbation on binding to the catalytic zinc ion in the enzyme . NAD' complex.Pyrazole is an effective inhibitor of the liver alcohol dehydrogenase reaction, strictly competitive with the alcohol substrate [l]. Theorell et al. demonstrated that pyrazole inhibition is attributable to the formation of an exceptionally firm enzyme . NAD' . pyrazole complex showing a characteristic difference absorption band at about 295 nm [2]. Due to the stability and chromophoric properties of this ternary complex, pyrazole has turned out to be a most useful ligand from a methodological point of view. Transient-state kinetic methods based on the absorbance changes associated with pyrazole binding have been applied to monitor formation of the enzyme . NAD' complex during catalysis and to study the process of ternary-complex formation with non-chromophoric substrate and coenzyme analogues [3-51. The tight binding of pyrazole in the ternary complex can be utilized to render NAD' dissociation insignificant in active-site titrations of the enzyme [2] and in kinetic studies of coenzyme binding [6]. The high affinity of the enzyme . NAD' complex for pyrazole makes it possible also to examine the kinetics of enzymic aldehyde reduction under single-turnover conditions by the 'catalytic suicide' method of McFarland and Bernhard [7].Despite the extensive use of pyrazole for such methodological purposes in experiments carried out at different pH, detailed kinetic information about pyrazole binding to the enzyme . NAD' complex is available only for the reaction at pH 7 [ 3 ] . The present investigation was undertaken to characterize the kinetics of the binding process over the wide pH range (4-10) usually considered in mechanistic stu...

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...

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.

1. Aldehyde binding to liver alcohol dehydrogenase in the absence and presence of coenzymes has been characterized by spectrometric equilibrium methods, using auramine 0 and bipyridine as reporter ligands.2. Free enzyme shows a significant affinity for aldehydes, and equilibrium constants for dissociation of the binary complexes formed with typical aldehyde substrates are reported. Binary-complex formation does not lead to any detectable inner-sphere coordination of aldehydes to the catalytic zinc ion of the enzyme subunit.3. Complex formation with NAD' or NADH increases the affinity of the enzyme for aromatic aldehydes by a factor of 1.8-3.5 and 6-17, respectively. Benzaldehyde and dimethylaminocinnamaldehyde binding to the enzyme . NAD ' complex is not detectably associated with inner-sphere coordination of the aldehyde to zinc.4. It is concluded that binding of NADH is required to induce catalytically adequate bonding interactions between enzyme and aromatic aldehydes. The effect of reduced coenzyme in this respect is attributed to hydrophobic interactions leading to dehydration ofthe active-site region, which allows aldehyde substrates to compete successfully with water for inner-sphere coordination to the catalytic zinc ion. Oxidized coenzyme is proposed to have a similar promoting effect on metal coordination of aldehydes which function as substrates for the dismutase activity of the enzyme.Liver alcohol dehydrogenase [I] catalyzes alcohol/aldehyde interconversion by a ternary-complex mechanism with NAD+/NADH as coenzymes. The enzymic reduction of aldehydes invariably has been found to be kinetically ordered with coenzyme binding preceding substrate binding [I -41. This could indicate that the enzyme shows an insignificantly low affinity for aldehyde substrates in the absence of NADH [5]. Apart from a preliminary (unconfirmed) report that acetaldehyde and butyraldehyde are bound in competition with the zinc-chelating reagent 2,2'-bipyridine [6], there is no direct evidence that aldehydes interact detectably with free enzyme. On the other hand, strong synergistic effects of coenzyme on the binding of substrate analogues have been documented in several instances [l, 51, so the affinity of liver alcohol dehydrogenase for aldehydes might well increase drastically on complex formation with NADH.

Complex formation at the general anion‐binding site of the liver alcohol dehydrogenase subunit has been characterized by transient‐state kinetic methods, using NADH as a reporter ligand. Equilibrium dissociation constants for anion binding at the site are reported. They conform basically to the lyotropic series of affinity order, with exceptionally tight binding of sulphate. The particular specificity for sulphate might be a general characteristic of anion‐binding enzymic arginyl sites. Anionic species of phosphate and pyrophosphate buffer solutions do not interact significantly with the general anion‐binding site over the pH range 8–10. At lower pH, phosphate binding becomes significant due to complex formation with the monovalent H2PO−4 species. The latter interaction corresponds to a dissociation constant of about 60 mM, indicating that phosphate binding is comparatively weak also at low pH. It is conluded that previously reported pH dependence data for coenzyme binding to liver alcohol dehydro‐ genase cannot be much affected by coenzyme‐competitive effects of buffer anion binding. Kinetic parameter estimates now determined for NADH binding in weakly buffered solutions agree within experimental precision with those obtained previously from measurements made in buffer solutions of 0.1 M ionic strength.