Synergism between Coenzyme and Aldehyde Binding to Liver Alcohol Dehydrogenase

Andersson, Pia; Kvassman, Jan; Oldén, Bertil; Pettersson, Gösta

doi:10.1111/j.1432-1033.1983.tb07513.x

Cited by 7 publications

(26 citation statements)

References 27 publications

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“…These kinetic data establish that the stabilizing effect of NADH on DACA binding [2] derives mainly from a decreased rate of dissociation of the aldehyde at the ternary-complex level.…”

Section: Daca Binding To the Enzymesupporting

confidence: 54%

“…These values can be taken as estimates of the rate constant for NADH dissociation from the respective complexes (k-l and k-4 in Scheme 1). The alternative possibility that weak or slow NAD+ binding may contribute significantly to rate-limitation of the displacement reactions is ruled out by binding data reported previously [2,17] and in Fig. 4.…”

Section: Effect Of Daca On Coenzyme Association Ratesmentioning

confidence: 95%

“…Three of the binding steps in Scheme 1 (NADH binding to free enzyme [I, 171, aldehyde binding to free enzyme [2], and aldehyde binding to the enzyme. NADH complex [16,20]) can be considered as essentially pH-independent between pH 6 and 9.…”

Section: Order Of Nadh and Daca Bindingmentioning

confidence: 99%

“…The enzyme shows a significant affinity for aldehyde substrates whether or not NADH is present (and vice versa) [1,2], which indicates that the productive enzyme. NADH .…”

mentioning

confidence: 99%

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Catalytic significance of binary enzyme‐aldehyde complexes in the liver alcohol dehydrogenase reaction

Andersson

Kvassman

Oldén

et al. 1984

European Journal of Biochemistry

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1. The interaction of liver alcohol dehydrogenase with NADH and aldehyde substrates has been characterized with respect to ternary-complex formation by the apparently non-preferred pathway which involves intermediate formation of binary enzyme . aldehyde complexes. Rate constant estimates are reported for dimethylaminocinnamaldehyde (DACA) binding to free enzyme and for NADH binding to the enzyme . DACA complex.2. The rate ofNADH (or NAD') association to liver alcohol dehydrogenaseis not detectably affected by DACA binding to the enzyme, but the NADH dissociation rate decreases approximately by a factor of 6. The NADHinduced increase in affinity of the enzyme for DACA is similarly attributable to a decreased dissociation rate rather than an increased association rate of the aldehyde. DACA dissociates much more rapidly than coenzyme from the enzyme. NADH . aldehyde complex and shows a higher association rate constant than NADH in its interaction with free enzyme.3. It is concluded from these results that the enzymic reduction of typical aldehyde substrates will conform to a rate equation which is experimentally indistinguishable from that of a compulsory-order mechanism with coenzyme binding preceding substrate binding, and that this rate equation will obtain irrespective of which pathway for ternary-complex formation is actually preferred. Rate equations provide no reliable information about the order of ligand binding in ternary-complex systems.4. A flow analysis is presented which indicates that coenzyme and substrate are actually bound in random order to liver alcohol dehydrogenase during the enzymic reduction of aldehydes by NADH. The enzyme. aldehyde pathway for ternary-complex formation is fully kinetically competent, and reaction flow via this pathway may predominate when aldehyde concentrations exceed those required for half-saturation of free enzyme. Binary enzyme . aldehyde complexes are seemingly insignificant with respect to the rate behaviour of the enzyme, but may provide most significant and even predominant contributions to the catalytic reaction flow.Liver alcohol dehydrogenase [I ] catalyzes alcohol/aldehyde interconversion by a ternary-complex mechanism with NAD+/NADH as coenzymes. The enzyme shows a significant affinity for aldehyde substrates whether or not NADH is present (and vice versa) [1,2], which indicates that the productive enzyme. NADH . aldehyde complexes can be assumed to be formed by a basically random-order binding mechanism (Scheme 1). The enzyme. aldehyde pathway for ternary-complex formation in Scheme 1 does not appear to contribute appreciably to the catalytic reaction flow, however. The enzymic reduction of aldehydes by NADH invariably has been found to conform to the rate equation of a compulsoryorder mechanism with coenzyme binding preceding binding of the aldehyde substrate [3 -61.The reason why this binding order appears to be preferred remains obscure. Essentially no information is available about the effect of coenzyme on the kinetics of substrate binding, or about th...

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“…These kinetic data establish that the stabilizing effect of NADH on DACA binding [2] derives mainly from a decreased rate of dissociation of the aldehyde at the ternary-complex level.…”

Section: Daca Binding To the Enzymesupporting

confidence: 54%

Section: Effect Of Daca On Coenzyme Association Ratesmentioning

confidence: 95%

Section: Order Of Nadh and Daca Bindingmentioning

confidence: 99%

“…The enzyme shows a significant affinity for aldehyde substrates whether or not NADH is present (and vice versa) [1,2], which indicates that the productive enzyme. NADH .…”

mentioning

confidence: 99%

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Catalytic significance of binary enzyme‐aldehyde complexes in the liver alcohol dehydrogenase reaction

Andersson

Kvassman

Oldén

et al. 1984

European Journal of Biochemistry

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“…Decanoate and benzoate are bound mainly through hydrophobic interactions at the substrate-binding site, and there is reason to believe that complex formation at this site may affect coenzyme binding independently of ligand charge [8]. The observed quantitative analogy between equilibrium constants for carboxylate and proton dissociation from different enzyme species, therefore, might be fortuitous or might reflect interactions which should not be related primarily to complex formation at the catalytic zinc ion.…”

mentioning

confidence: 99%

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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Characterization of recombinant xylitol dehydrogenase from Galactocandida mastotermitis expressed in Escherichia coli

Nidetzky

Helmer

Klimacek

et al. 2003

Chemico-Biological Interactions

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Synergism between Coenzyme and Aldehyde Binding to Liver Alcohol Dehydrogenase

Cited by 7 publications

References 27 publications

Catalytic significance of binary enzyme‐aldehyde complexes in the liver alcohol dehydrogenase reaction

Catalytic significance of binary enzyme‐aldehyde complexes in the liver alcohol dehydrogenase reaction

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

Characterization of recombinant xylitol dehydrogenase from Galactocandida mastotermitis expressed in Escherichia coli

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