Anion Binding to Liver Alcohol Dehydrogenase

Oldén, Bertil; Pettersson, Gösta

doi:10.1111/j.1432-1033.1982.tb06684.x

Cited by 21 publications

(21 citation statements)

References 24 publications

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“…5). The latter rate parameter should be competitively affected by complex formation at the general anion-binding site [9], and data in Fig. 7 confirm that such effects are at hand with a coenzyme-competitive ligand such as SCN-.…”

Section: Binding Site For Cyanidesupporting

confidence: 54%

“…The present investigation was undertaken to obtain such information. Data now reported seem to establish that cyanide (in contrast to a large number of inorganic anions previously examined [9]) combines to catalytic zinc on complex formation with liver alcohol dehydrogenase. We have, therefore, characterized the synergism between coenzyme and cyanide binding to the enzyme by equilibrium measurements and transient-state kinetic methods.…”

mentioning

confidence: 66%

“…Depending on the length of the side-chain, ligation to catalytic zinc may (decanoate [6]) or may not (formate, acetate [9]) predominate in the binary enzymic complexes formed with fatty acids. Binding data obtained in our laboratory indicate that the metal site is preferred by propionate and higher homologues.…”

Section: Synergism Between Coenzyme and Anion Bindingmentioning

confidence: 99%

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

Andersson¹,

Kvassman²,

Oldén³

et al. 1984

European Journal of Biochemistry

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1. The synergism between coenzyme and anion binding to liver alcohol dehydrogenase has been examined by equilibrium measurements and transient-state kinetic methods to characterize electrostatic interactions of coenzymes with ligands which are bound to the catalytic zinc ion of the enzyme subunit.2. Inorganic anions typically exhibit an at least 200-fold higher affinity for the general anion-binding site than for catalytic zinc on complex formation with free enzyme. Acetate and SCN -interact more strongly with catalytic zinc in the enzyme. NAD' complex than with the general anion-binding site in free enzyme. CN-shows no significant affinity for the general anion-binding site, but combines to catalytic zinc in the absence as well as the presence of coenzymes.3. Coordination of CN-to catalytic zinc weakens the binding of NADH by a factor of 50, and tightens the binding of NAD' to approximately the same extent through interactions which do not include any contributions from covalent adduct formation between CN-and NAD'. These observations provide unambiguous information about the magnitude of electrostatic field effects of coenzymes on anion (e.g. hydroxyl ion) binding to catalytic zinc. They lead to the important inference that coenzyme binding must be strongly affected by ionization of zinc-bound water irrespective of the actual acidity of the latter group.4. It is concluded on such grounds that the much debated pH dependence of coenzyme binding to liver alcohol dehydrogenase must derive from ionization of zinc-bound water. The assumption that such is not the case leads to the inference that there is no detectable effect of ionization of zinc-bound water on coenzyme binding over the p H range 6-12, a possibility which is definitely excluded by the present results.The effect of pH on the catalytic activity of liver alcohol dehydrogenase has been extensively studied and intensively discussed [I, 21. 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]. This would require that ionization of zinc-bound water increases (decreases) the affinity of the enzyme for NAD' (NADH) by a factor of 40 (100).Evidence suggesting that zinc-bound anions may give rise to electrostatic field effects of that order of magnitude has been obtained by examination of' the synergism between coenzyme binding and the binding of negatively charged substrate analogues such as decanoate and benzoate [5,6]. Complex formation between enzyme and the latter carboxylates was found to result in a 40-fold stabilization of N A D + binding and a slightly lar...

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Section: Binding Site For Cyanidesupporting

confidence: 54%

mentioning

confidence: 66%

Section: Synergism Between Coenzyme and Anion Bindingmentioning

confidence: 99%

See 1 more Smart Citation

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

Andersson¹,

Kvassman²,

Oldén³

et al. 1984

European Journal of Biochemistry

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“…The saturation curve obtained at pH 8.5 could not be satisfactory fitted with eq 5 used earlier (Oldén & Pettersson, 1982). …”

Section: Evaluation Of Rapid Kineticmentioning

confidence: 74%

“…Most earlier studies were performed in phosphate buffers which were shown to be coenzyme-competitive at low pH (Oldén & Pettersson, 1982). In this investigation the presence of any significant concentration of anions, able to interfere with the coenzyme binding process, was strictly avoided.…”

Section: Reasons Why the New Set Of Constants Differs From Earlier Pumentioning

confidence: 99%

Electrostatic Effects in the Kinetics of Coenzyme Binding to Isozymes of Alcohol Dehydrogenase from Horse Liver

1997

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The kinetic mechanism for the binding of NAD+ and NADH to the EE and SS isozymes of alcohol dehydrogenase (LADH) was studied between pH 7 and pH 10 by monitoring the quenching of tryptophan fluorescence. A consistent interpretation of all data was only possible by introducing a two-step binding mechanism. The first binding step is related to docking of the adenosine part of the coenzymes and the subsequent isomerization to the binding of the nicotinamide part. At high NADH concentrations an additional slow isomerization was identified as a conformational transition of the protein. A pH dependence for NADH binding is observed which is restricted to changes in the binding kinetics of the adenosine moiety going from pH 7 to pH 10, a tendency which is similar also for NAD+. This is attributed to pH-dependent variations in electrostatic attractions acting as a steering force of the docking process. The nicotinamide docking of NADH is equally fast for both isozymes and pH-independent over the measured range, whereas this docking equilibrium for NAD+ is pH-dependent for EE- and SS-LADH alike and the rate of association comparable. Presumably, a GluEE-366-LysSS substitution results in a stronger binding and faster association of both oxidized and reduced cofactor to the SS isozyme. A structural proof is presented for coenzyme-competitive binding of a sulfate ion, resulting in electrostatic shielding.

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The influence of anions and inhibitors on the catalytic metal ion in Co(II)-substituted horse liver alcohol dehydrogenase

et al. 1987

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1H-NMR and electronic spectroscopic data are reported for the interaction of the effector molecule imidazole and the inhibitor molecule pyrazole with horse liver alcohol dehydrogenase whose catalytic zinc ions were replaced by Co(II). In addition 13C-NMR and optical data are given for the binding of acetate to this enzyme species. For the binary complex with imidazole an assignment of the protons of the metal-coordinated imidazole has been made and it was found that the rate of exchange of the effector molecule is slow on the NMR time scale. In the presence of NADH which is bound to the open conformation of the binary complex, the most pronounced change is a shift of the beta-CH2 protons of the metal-coordinated cysteine residues which is attributed to hydrogen bonding interactions between the carboxamide group of the nicotinamide moiety with cysteine 46. The 1H-NMR spectra of the binary complex of Co(II)-HLADH with pyrazole show resonances assigned to the protons in the 3- and 4-positions of the bound inhibitor, the NH proton resonance is not detectable. In the ternary complex with pyrazole and NAD+ only the resonances of the beta-CH2 protons (beyond 150 ppm) are changed whereas the protons of histidine 67 and the bound inhibitor are unchanged. The data demonstrate that the coordination environment of the catalytic metal ion is changed very little when the protein changes from the open to the closed conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

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Anion Binding to Liver Alcohol Dehydrogenase

Cited by 21 publications

References 24 publications

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

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

Electrostatic Effects in the Kinetics of Coenzyme Binding to Isozymes of Alcohol Dehydrogenase from Horse Liver

The influence of anions and inhibitors on the catalytic metal ion in Co(II)-substituted horse liver alcohol dehydrogenase

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