The structure of horse liver alcohol dehydrogenase

Eklund, Hans; Nordström, B.; Zeppezauer, E.; Söderlund, Gustaf; Ohlsson, Ingrid; Boiwe, Torne; Brändén, Carl‐Ivar

doi:10.1016/0014-5793(74)80725-8

Cited by 188 publications

(103 citation statements)

References 7 publications

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“…Furthermore a large number of solution studies [23] have demonstrated the existence of a hydrophobic binding region for large substrate molecules, which is consistent with the fact that this pocket is lined almost exclusively by hydrophobic residues [l]. The present investigation confirms the mechanism of action previously suggested from a correlation of crystallographic and solution studies [23,30]. This mechanism is based on the crystallographic facts that the only polar groups in the catalytic region are Ser-48, Thr-178, zinc and the zinc-bound water molecule.…”

Section: Discussionsupporting

confidence: 91%

X‐Ray Investigation of the Binding of 1,10‐Phenanthroline and Imidazole to Horse‐Liver Alcohol Dehydrogenase

Boiwe

Brändén

1977

European Journal of Biochemistry

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We have studied the binding of two inhibitor molecules, imidazole and 1 ,lo-phenanthroline, to liver alcohol dehydrogenase by crystallographic methods. X-ray data for the imidazole complex were collected to 0.29-nm resolution and for the 1,lO-phenanthroline complex to 0.45-nm resolution. In both cases we found only one peak in the difference electron density maps close to the active zinc atom. The peak corresponding to 1,lO-phenanthroline overlaps the site of the density of the zinc-bound water in the apoenzyme and the imidazole density partly overlaps this density. We can not discern any additional peaks close to the zinc atom which would correspond to new positions of bound water. We thus conclude that both these inhibitors bind to the catalytic zinc atom and that upon binding they displace the water molecule that is firmly bound to this zinc atom in the apoenzyme. We do not see any structural changes in the remaining part of the molecule.The three-dimensional structure of horse liver alcohol dehydrogenase is known to high resolution [l]. It is thus now possible to try to elucidate the mechanism of action of this enzyme by crystallographic studies of the binding of suitably chosen inhibitor molecules for both coenzyme and substrates. Details of coenzyme binding have been deduced in this way in several studies using both coenzyme analogues [2 -41, which bind isomorphously to the orthorhombic apoenzyme structure, and a combination of NADH and inhibitor [5], which produces triclinic holoenzyme crystals. These studies confirmed earlier observations [6,7] that the zinc atom, which is necessary for catalytic action, is not directly involved in coenzyme binding. We here report studies of complexes with inhibitor molecules that bind directly to the catalytically active zinc atoms and thus in all probability define the binding areas of substrate molecules. Two such complexes have been studied using the inhibitor molecules 1 ,lo-phenanthroline and imidazole.It towards both coenzyme and substrate but a careful analysis [13,14] demonstrated that this kinetic behaviour could be attributed solely to the formation of an inactive binary enzyme . 1,lO-phenanthroline complex. Using double difference spectrophotometry Yonetani [15,16] could show that this large inhibitor molecule competed with the nicotinamide moiety of the coenzyme and demonstrated the existence of a ternary complex between enzyme, ADP-ribose and 1,lO-phenanthroline. Further solution evidence that this inhibitor binds in the substrate and nicotinamide binding areas has been provided by Sigman [17], who has shown competition between binding of the similar inhibitor 2,2-bipyridine and substrate molecules.Imidazole shows a different inhibition pattern. Theorell and McKinley McKee [18,19] have demonstrated the existence not only of a binary enzyme . imidazole complex but also of inactive ternary complexes with both oxidized and reduced coenzyme. Furthermore they have also shown [18] that imidazole abolishes the pH dependence of NAD+ binding, and thus interferes...

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Section: Discussionsupporting

confidence: 91%

X‐Ray Investigation of the Binding of 1,10‐Phenanthroline and Imidazole to Horse‐Liver Alcohol Dehydrogenase

Boiwe

Brändén

1977

European Journal of Biochemistry

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“…Catalytic Zn'+ centres have 3 stronglybound protein ligands (3 His in carbonic anydrase; 2 His, 1 Glu in carboxypeptidase A and thermolysin; and 2 Cys, 1 His in liver alcohol dehydrogenase) and the site 4 is available to the substrate through Hz0 displacement. The structural Zn*+ ion in liver alcohol dehydrogenase, on the other hand, occupies a Zn(Cys)4 site [7] . Although the electron micrograph of metallothionein in [8] was interpreted in terms of Cd& centres, there is considerable overlap of most of their neighbouring coordination triangles as would be expected if bridging sulphurs are present.…”

Section: Resultsmentioning

confidence: 99%

¹¹³Cd nuclear magnetic resonance of metallothionein

Sadler¹,

Bakka²,

Beynon³

1978

FEBS Letters

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“…The interactions between the adenosine moiety of the nucleotide and the enzymc have been described earlier [8]. The position of the phosphate group of AMP is in a cleft in the border region between the two domains [8] of the subunit.…”

Section: Tip Eiiz~ww a M P Coiiiplesmentioning

confidence: 92%

“…The position of the phosphate group of AMP is in a cleft in the border region between the two domains [8] of the subunit. The residue that seems to be mainly responsible for binding this group is Arg-47.…”

Section: Tip Eiiz~ww a M P Coiiiplesmentioning

confidence: 99%

Carboxymethylation of Horse‐Liver Alcohol Dehydrogenase in the Crystalline State

Zeppezauer

Ohlsson

Jörnvall

1975

European Journal of Biochemistry

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Horse liver alcohol dehydrogenase (isozyme EE) in the crystalline state was alkylated with iodoacetate under conditions resulting in the single substitution of Cys-46, which is a ligand to the active-site zinc atom. Alkylation was facilitated by the prior formation of a complex with imidazole bound to the zinc atom. Extent and specificity of the reaction were determined by use of 14C-labelled iodoacetate and by analyses of radioactive peptides after cleavage with trypsin. Ternary complexes of the enzyme with coenzymes and inhibitors effectively protected the protein against alkylation. ADP-ribose, Pt(CN)i-, I,lO-phenanthroline, Au(CN); and AMP also prevented alkylation with decreasing effectiveness.Crystallographic studies of the alkylated enzyme show that the carboxymethylated sulfur atom of Cys-46 is still liganded to the active-site zinc atom and that theiodide ion liberated during alkylation is bound as the fourth ligand to zinc, displacing imidazole. Crystallographic analyses were also performed of the binding of AMP and Pt(CN)i-to the enzyme. It was found that Arg-47 interacts with the phosphate moiety of the nucleotide. Lys-228 and Arg-47 interact in the platinate complex with the bulky anion, the center of which coincides with the position of the nucleotide phosphate. Some of the cyano-ligands to platinum occupy a crevice between the coenzyme phosphate binding site and the active-site zinc atom.The results of the combined studies on primary and tertiary structures confirm previous suggestions that iodoacetate enters the active site via reversible binding to an anion-binding site. This site interacts with the negatively charged groups of the coenzyme as well as with ADP-ribose, Pt(CN):-and to a lesser extent Au(CN); and AMP, which therefore prevent the reversible binding of iodoacetate. 1,lO-Phenanthroline does not block the binding site but interferes with alkylation presumably by changing the coordination of zinc.Identification of this labelled residue in both chemical and crystallographic studies correlates the primary and tertiary structures. Characterizations of the active-site zinc region and the general anion-binding site are also presented.

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The structure of horse liver alcohol dehydrogenase

Cited by 188 publications

References 7 publications

X‐Ray Investigation of the Binding of 1,10‐Phenanthroline and Imidazole to Horse‐Liver Alcohol Dehydrogenase

X‐Ray Investigation of the Binding of 1,10‐Phenanthroline and Imidazole to Horse‐Liver Alcohol Dehydrogenase

¹¹³Cd nuclear magnetic resonance of metallothionein

Carboxymethylation of Horse‐Liver Alcohol Dehydrogenase in the Crystalline State

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The structure of horse liver alcohol dehydrogenase

Cited by 188 publications

References 7 publications

X‐Ray Investigation of the Binding of 1,10‐Phenanthroline and Imidazole to Horse‐Liver Alcohol Dehydrogenase

X‐Ray Investigation of the Binding of 1,10‐Phenanthroline and Imidazole to Horse‐Liver Alcohol Dehydrogenase

113Cd nuclear magnetic resonance of metallothionein

Carboxymethylation of Horse‐Liver Alcohol Dehydrogenase in the Crystalline State

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¹¹³Cd nuclear magnetic resonance of metallothionein