Mechanisms of zinc ion catalysis in small molecules and enzymes

Dunn, Michael F.

doi:10.1007/bfb0116550

Cited by 71 publications

(33 citation statements)

References 169 publications

(125 reference statements)

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“…As predicted by theory, the exponential transient governing this relaxation process was found to conform to Eqns (2)(3)(4). The typical results in Fig.…”

Section: Nadh Bindingsupporting

confidence: 56%

“…By analogy to the catalytic zinc ion in liver alcohol dehydrogenase [l], the active-site zinc ion in carboxypeptidase and carbonic anhydrase is bound by three protein ligands and coordinates a water molecule or hydroxyl ion as a fourth inner-sphere ligand [2]. Spectroscopic and kinetic evidence has been presented which indicates that the p& of zinc-bound water in carbonic anhydrase is about 7 [18].…”

Section: Eflect Ojligand Field On the Pand Of Zinc-bound Watermentioning

confidence: 99%

“…Spectroscopic and kinetic evidence has been presented which indicates that the p& of zinc-bound water in carbonic anhydrase is about 7 [18]. This value has been considered as unreasonably low in comparison to the p& values observed for aquo ligands in six-coordinate zinc complexes [2]. A decrease in coordination number of the metal, however, would be expected to increase the acidity of metal-bound water.…”

Section: Eflect Ojligand Field On the Pand Of Zinc-bound Watermentioning

confidence: 99%

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Effect of NADH on the pK_a of Zinc‐Bound Water in Liver Alcohol Dehydrogenase

Andersson

Kvassman

Lindström

et al. 1981

European Journal of Biochemistry

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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.

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“…As predicted by theory, the exponential transient governing this relaxation process was found to conform to Eqns (2)(3)(4). The typical results in Fig.…”

Section: Nadh Bindingsupporting

confidence: 56%

Section: Eflect Ojligand Field On the Pand Of Zinc-bound Watermentioning

confidence: 99%

Section: Eflect Ojligand Field On the Pand Of Zinc-bound Watermentioning

confidence: 99%

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Effect of NADH on the pK_a of Zinc‐Bound Water in Liver Alcohol Dehydrogenase

Andersson

Kvassman

Lindström

et al. 1981

European Journal of Biochemistry

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“…The affinity increase caused by NADH binding may be of physiological importance as a partial determinant of the K, value for aldehyde substrates, but there is otherwise little reason to emphasize the mechanistic implications of this synergistic effect. In particular, the idea must be rejected that the synergism between coenzyme and substrate binding conveys a discernable kinetic order to their binding during catalysis [5]. If NADH binding stabilizes complex formation with aldehydes to a certain extent, then aldehyde binding must stabilize complex formation with NADH to exactly the same extent and the net effect on the order of catalytic NADH/aldehyde binding will be nil.…”

Section: Effect Qf Coenzymes On Aldehyde Bindingmentioning

confidence: 99%

Synergism between Coenzyme and Aldehyde Binding to Liver Alcohol Dehydrogenase

Andersson¹,

Kvassman²,

Oldén³

et al. 1983

European Journal of Biochemistry

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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.

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“…liver alcohol dehydrogenase is likely to have a physiological function which requires effective catalysis of both aldehyde reduction and alcohol oxidation. With NADH/NAD+ as the coenzymes, the thermodynamic equilibrium for alcohol/ aldehyde interconversion is greatly in Favour of aldehyde reduction at physiological pH [31]. This means that liver alcohol dehydrogenase would be expected to be designed primarily to facilitate the thermodynamically unfavourable process of alcohol oxidation.…”

Section: Tlzc Cntcilytic Functioti O F Thc Active-site Zinc Ionmentioning

confidence: 99%

Substituent Effects on the Ionization Step Regulating Desorption and Catalytic Oxidation of Alcohols Bound to Liver Alcohol Dehydrogenase

Kvassman¹,

Larsson²,

Pettersson³

1981

Eur J Biochem

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1. Transient-state kinetic methods, based on the use of pyrazole as a displacing reagent and reporter ligand, have been applied to examine the pH dependence of rate and equilibrium constants for 2-chloroethanol and 2,2-dichloroethanol binding to the binary complex formed between liver alcohol dehydrogenase and NAD' .2. The apparent affinity of the enzyme . NAD' complex for the examined alcohols is dependent on two proton dissociation equilibria. One of these equilibria affects the rate of alcohol association to the binary complex with a ligand-independent pKa value of 7.6, attributable to ionization of zinc-bound water in the enzyme. NAD' complex. The second proton dissociation equilibrium regulates the rate of alcohol desorption from the enzyme . NAD' . alcohol complex and exhibits a pK, value of 5.4 and 4.5, respectively, with 2-chloroethanol and 2,2'-dichloroethanol as the ligand. Steady-state kinetic data are reported which indicate that the pK,-5.4-equilibrium controls also the apparent rate of enzymic hydride transfer from 2-chloroethanol to NAD '. 3. These results lend strong support to the mechanism of enzyme action proposed by Kvassman andPettersson according to which the enzyme . NAD' . alcohol complex participates in an obligatory proton dissociation step which regulates both desorption and catalytic oxidation of the alcohol ligand. The corresponding pKa value is shown to be lineary dependent on the pK, of the free alcohol ligand with a regression coefficient (Bronstedt a value) of about 0.6. The latter observation provides direct evidence that the effect of pH on the reactivity of the enzyme . NAD' . alcohol complex reflects an alcohol/alcoholate ion equilibration of the enzyme-bound substrate. 4. The particular mode of binding and properties of the active-site zinc ion in liver alcohol dehydrogenase suggest that the catalytic function of the metal ion can be related primarily to facilitation of the process of alcohol oxidation through facilitation of the alcoholate ion formation step now established to precede hydride transfer to NAD' Liver alcohol dehydrogenase catalyzes the oxidation of alcohols by NAD'. The enzyme operates by an effectively ordered ternary-complex mechanism (Scheme l), coenzyme binding preceding binding of the alcohol substrate [l]. Investigations of the effect of pH on the catalytic steps of alcohol binding [2,3] and ternary-complex interconversion [4,5] have shown that the enzyme . NAD' . alcohol complex participates in a proton dissociation equilibrium which regulates the reactivity of this reaction intermediate. Alcohol desorption occurs exclusively from the protonated form of the ternary complex, while hydride transfer from the alcohol substrate to NAD' requires the unprotonated form of the complex.It has been suggested in previous reports from our laboratory [3,5] that the effect of pH on the reactivity of the enzyme . NAD+ . alcohol complex derives from an alcohol/ alcoholate ion equilibration of the enzyme-bound substrate (Scheme 2). Determinations of the corresponding ac...

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Mechanisms of zinc ion catalysis in small molecules and enzymes

Cited by 71 publications

References 169 publications

Effect of NADH on the pK_a of Zinc‐Bound Water in Liver Alcohol Dehydrogenase

Effect of NADH on the pK_a of Zinc‐Bound Water in Liver Alcohol Dehydrogenase

Synergism between Coenzyme and Aldehyde Binding to Liver Alcohol Dehydrogenase

Substituent Effects on the Ionization Step Regulating Desorption and Catalytic Oxidation of Alcohols Bound to Liver Alcohol Dehydrogenase

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Mechanisms of zinc ion catalysis in small molecules and enzymes

Cited by 71 publications

References 169 publications

Effect of NADH on the pKa of Zinc‐Bound Water in Liver Alcohol Dehydrogenase

Effect of NADH on the pKa of Zinc‐Bound Water in Liver Alcohol Dehydrogenase

Synergism between Coenzyme and Aldehyde Binding to Liver Alcohol Dehydrogenase

Substituent Effects on the Ionization Step Regulating Desorption and Catalytic Oxidation of Alcohols Bound to Liver Alcohol Dehydrogenase

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Effect of NADH on the pK_a of Zinc‐Bound Water in Liver Alcohol Dehydrogenase

Effect of NADH on the pK_a of Zinc‐Bound Water in Liver Alcohol Dehydrogenase