The Structure of Horse-Liver Alcohol Dehydrogenase. Iii. Molecular Structure at 5 Å Resolution

Brändén, Carl‐Ivar; Zeppezauer, E.; Söderberg, Bengt-Olof; Boive, T.; Nordström, B.; Söderlund, Gustaf; Zeppezauer, Michael; Werner, P.; Åkeson, Åke

doi:10.1016/b978-0-08-016874-6.50017-5

Cited by 9 publications

(8 citation statements)

References 18 publications

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“…Crystals of native enzyme were prepared according to procedures described earlier [24]. The complex with 1 ,lo-phenanthroline was prepared by dialysing native crystals for 2 days in a solution of 0.01 M Tris/HCl buffer and 0.005 M 1,lO-phenanthroline at pH 8.5 containing 25 % v/v 2-methyl-2,4-pentanediol.…”

Section: Methodsmentioning

confidence: 99%

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: Methodsmentioning

confidence: 99%

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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“…Crystals of the enzyme were prepared according to the procedure described earlier [7]. These enzyme crystals were first used to prepare binary enzyme.…”

Section: Methodsmentioning

confidence: 99%

The Crystal Structure of Complexes between Horse Liver Alcohol Dehydrogenase and the Coenzyme Analogues 3‐Iodopyridine‐adenine Dinucleotide and Pyridine‐adenine Dinucleotide

Samama

Zeppezauer

Biellmann

et al. 1977

European Journal of Biochemistry

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We have studied the binding of the enzymatically active NAD + analogue, 3-iodopyridine-adenine dinucleotide, and the inactive analogue, pyridine-adenine dinucleotide to the enzyme horse liver alcohol dehydrogenase using X-ray crystallographic methods. These studies were made under such conditions that crystals of the complexes were isomorphous to apocnzyme crystals. Both analogues bind in the same conformation. The binding of the adenosine moiety is very similar to that of ADP-ribose or NADH bound to the enzyme. The conformation and mode of binding of the remaining portions of the analogue molecules is, however, quite different. The pyridine ring is not situated in the active-site pocket as the nicotinamide group in thc isomorphous enzyme -NADH . imidazole complex but lies at the surface of the crevice between the two domains of the subunit, approximately 1.5 nm away from the catalytically active zinc atom. Lys-228 which has been shown to be important for NADH dissociation is in this region of the molecule.A large number of analogues to the coenzyme NAD' have been investigated [l] in order to delineate the role of the various moieties of the dinucleotide in the interaction of this coenzyme with the NAD+-dependent dehydrogenases. We have been particularly interested in the enzymatic effects of substitutions on the nicotinamide ring. As part of these studies, and also for thc use as heavy-atom derivatives in protein Crystallography, we synthesized 3-iOdO and subsequently 3-bromo and 3-chloro derivatives of pyridineadenine dinucleotide which were active in enzymatic hydrogen transfer reactions. Synthesis of these analogues and their kinetic properties for some NAD+-dependent dehydrogenase-catalysed reactions is presented elsewhere [2].The present study describes the binding of the 3-iodo analogue to one of these enzymes, horse liver alcohol dehydrogenase, as studied by X-ray crystallographic methods. Furthermore, the results of that Abbreviations.Pd AD + . pyridine-adenine dinucleot ide ; io3PdAD+, 3-iodopyridine-adenine dinucleotide; Tes, 2-([tris-(hydroxymethyl)methyi]amino)ethane sulfonic acid.

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“…From our 5-A work we already knew the position of one of these zinc atoms and the subunit Biochemistry: BrAnd6n et al boundaries (11). The remaining high density within one subunit could then easily be allocated to one continous chain with corresponding side-chain densities.…”

Section: Conformation Of the Subunitmentioning

confidence: 99%

“…Adams et al (19) have sug- gested that the main "trigger" for the change of structure in LDH is an interaction between the adenosine phosphate and an arginine residue within this loop. Since a corresponding loop is absent in LADH, its conformational change must be induced by some other mechanism which in turn must involve the nicotinamide part of the coenzyme (11).…”

Section: Functional Significance Of the Structurementioning

confidence: 99%

Structure of Liver Alcohol Dehydrogenase at 2.9-Å Resolution

Brändén¹,

Eklund²,

Nordström³

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

175

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The conformation of the polypeptide chain in horse liver alcohol dehydrogenase (EC 1.1.1.1), as well as the binding sites for some inhibitor molecules, have been determined from x-ray crystallographic data to a resolution of 2.9 A. The apoenzyme of LADH crystallizes in space-group C2221 with one subunit per asymmetric unit and cell dimensions a = 56.0 A, b = 75.2 A, and c = 181.6 A (10). The crystallographic 2-fold axis relating the two subunits of the apoenzyme molecule is not present in crystals of complexes between apoenzyme and coenzyme, which suggests that the coenzyme may induce a structural asymmetry in the chemically identical subunits. An electron density map to 5-A resolution of the apoenzyme molecule has been described (11).Here we report the structure of this molecule as deduced from an electron-density distribution at a resolution of 2.9 A. Most important is the analysis of the binding sites for some inhibitor molecules, which permits us to identify functional attributes of the enzyme structure; MATERIALS AND METHODSThe enzyme was isolated from fresh horse livers (Akeson, A. & Lundqvist, G., to be published). Preparation of crystals and heavy-atom derivatives suitable for x-ray studies has been described (10). Methods of isomorphous replacement similar to those used for other protein-structure determinations were applied to obtain the electron density map. The crystallographic data were measured at +40 on a computercontrolled Philips-Stoe four-circle diffractometer equipped with a 32 K disc storage. Data were collected to a resolution of 2.9 X from crystals of the native protein, three heavy-metal derivatives [K2Pt(CN)4, KAu(CN)2, and K2Pt(CN)4 + KAu(CN)2], and one inhibitor complex [adenosine diphosphate ribose (ADP-ribose) ]. Intensities within 4.5 A were also measured on two other inhibitor complexes: 8-Br-ADPribose and 1,10-phenanthroline. A skeletal model of the main chain was built with the Kendrew-type models, with an optical comparator (12). CONFORMATION OF THE SUBUNITIn this section we describe briefly the conformation of the polypeptide chain and some details of the subunit interaction and binding of inhibitor molecules as deduced from our electron-density maps. In the next section we will discuss some implications of this structure.The two highest features in our 2.9-A electron-density map were roughly spherical in shape and were interpreted as the two zinc atoms of the subunit. From our 5-A work we already knew the position of one of these zinc atoms and the subunit

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The Structure of Horse-Liver Alcohol Dehydrogenase. Iii. Molecular Structure at 5 Å Resolution

Cited by 9 publications

References 18 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

The Crystal Structure of Complexes between Horse Liver Alcohol Dehydrogenase and the Coenzyme Analogues 3‐Iodopyridine‐adenine Dinucleotide and Pyridine‐adenine Dinucleotide

Structure of Liver Alcohol Dehydrogenase at 2.9-Å Resolution

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