Evidence for two different complexes of liver alcohol dehydrogenase with imidazole

Reynolds, C. Hugh; McKinley-McKee, John S.

doi:10.1016/0014-5793(72)80187-x

Cited by 8 publications

(5 citation statements)

References 9 publications

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“…Pyrazole is also found to bind directly to zinc in both the binary complex and the ternary complexes. Direct binding to zinc of imidazole and pyrazole is also in agreement with solution studies using a number of different methods (Theorell & McKinley-McKee, 1961;Theorell & Yonetani, 1963;Theorell et al, 1969;Shore & Gilleland, 1970;Reynolds & McKinley-McKee, 1972;Andersson, P., et al, 1981;Andersson, I., et al, 1981;Makinen & Yim, 1981). Contradictory conclusions have, however, been reported from NMR experiments with metal-sustituted enzymes.…”

Section: Discussionsupporting

confidence: 76%

Pyrazole binding in crystalline binary and ternary complexes with liver alcohol dehydrogenase

Eklund¹,

Samama²,

Wallen³

1982

Biochemistry

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Pyrazole is a strong inhibitor of liver alcohol dehydrogenase in combination with oxidized coenzyme NAD+. We have studied three different complexes of the inhibitor with the enzyme by using crystallographic methods: (1) the binary complex with pyrazole to 3.2-A resolution, (2) the ternary ternary complex with NAD+-4-iodopyrazole to 2.9-A resolution. Crystals of the binary complex are isomorphous to the apoenzyme, and pyrazole binds to the active-site zinc atom in a way analogous to imidazole. Crystals of the two ternary complexes are isomorphous with the ternary alcohol dehydrogenase-NADH-dimethyl sulfoxide complex. One of the nitrogen atoms of the pyrazole ring is directly bound to the active-site zinc atom with a Zn-N bond distance of 2.1A. The other nitrogen atom is 2 A from the C4 atom of the nicotinamide ring of the coenzyme. The iodine atom in 4-iodopyrazole is located in the hydrophobic substrate cleft. The effect of substitutions on the pyrazole ring are discussed in relation to the structure of the active site and substrate pocket. Pyrazole derivatives with long alkyl chains bound in the 4 position are outstanding inhibitors, and this property is related to the topography of the hydrophobic substrate cleft. The conformation of the oxidized coenzyme in the ternary complexes is essentially the same as that of the reduced coenzyme NADH in the NADH-dimethyl sulfoxide complex.

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Section: Discussionsupporting

confidence: 76%

Pyrazole binding in crystalline binary and ternary complexes with liver alcohol dehydrogenase

Eklund¹,

Samama²,

Wallen³

1982

Biochemistry

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“…The present conclusion that iinidazole binding to free enzyme exhibits a pk', 9.2 dependence analogous to that of the binding of NADH and zinc-chelating reagents seems to be consistent with and supported by previously reported data for binary-complex formation with imidazole at different pH [5,8,20]. For example, Reynolds et al pointed out that the binding of imidazole and 1 ,lo-phenanthroline is sevenfold weaker at pH 10 than at about pH 7 [2O].…”

Section: Binding Mechanism Jor Complex Formution At the Catalytic Zinsupporting

confidence: 78%

“…Anionic ligands were added in concentrations high enough to give an equilibrium solution containing more than 90% of the enzyme in the form of a ternary enzyme inhibitor. anion complex {0.4mM NADH [5,8], 4mM ADP-ribose [12], or 10 mM Pt(CN)i-[2]}. The assumption was made (and is justified by the present…”

Section: Titrimtjtvic Experimentsmentioning

confidence: 82%

“…However that may be, the observation that imidazole abolishes the pK, 7.6 dependence of NAD' dissociation [5] establishes that the ionization properties of zinc-bound water are drastically affected by imidazole binding. Our attention, therefore, has been drawn to the report that imidazole does not abolish the pH dependence of NADH binding [8]. A confirmation of the latter result would provide conclusive evidence that the pK, 9.2 group cannot be identical with zinc-bound water.…”

Section: Identity Of the Ionizing Group Rtjgulating Coenzyme Associationmentioning

confidence: 84%

“…The observation that there is no effect of pH on imidazole binding to the enzyme . NADH complex [5,8] indicates that the pKa 9.2 dependence of ligand binding to zinc is similarly abolished by NADH. Furthermore, titrimetric experiments have provided direct evidence that the pK, of the ionizing group controlling coenzyme association and anion binding is perturbed from 9.2 to a value above 10 in the binary complexes formed with NADH, AMP and…”

Section: Interrelutionship Between Lonizution Of Zinc-bound Wuter Undmentioning

confidence: 99%

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

Andersson¹,

Kvassman²,

Lindström³

et al. 1980

European Journal of Biochemistry

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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...

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Carboxymethyl horse-liver alcohol dehydrogenase

Reynolds

McKinley-McKee

1975

Archives of Biochemistry and Biophysics

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Evidence for two different complexes of liver alcohol dehydrogenase with imidazole

Cited by 8 publications

References 9 publications

Pyrazole binding in crystalline binary and ternary complexes with liver alcohol dehydrogenase

Pyrazole binding in crystalline binary and ternary complexes with liver alcohol dehydrogenase

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

Carboxymethyl horse-liver alcohol dehydrogenase

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