Crystallization of Horse Liver Alcohol Dehydrogenase Complexes from Alcohol Solutions.

Zeppezauer, E.; Söderberg, Bengt-Olof; Brändén, Carl‐Ivar; Åkeson, Åke; Theorell, Hugo; Blinc, R.; Paušak, S.; Ehrenberg, L.; Dumanović, J.

doi:10.3891/acta.chem.scand.21-1099

Cited by 61 publications

(23 citation statements)

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“…Although the limiting PKa of 5.0 was observed with the acetimidylated enzyme, we, think that the native enzyme must have the corresponding pKa with a somewhat lower value. The modified enzyme is, after all, a quite respectable catalyst for alcohol dehydrogenation, being 2.6 times more active than the native enzyme at pH 8 (Zoltobrocki et al, 1974 (Zeppezauer et al, 1967), it is reasonable and most simple to interpret the scheme as showing conformational equilibria. Then the high-fluorescence form would correspond to the orthorhombic conformation and the lowfluorescence form to the triclinic conformation.…”

Section: Discussionmentioning

confidence: 99%

pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases

Parker¹,

Hardman²,

Plapp³

et al. 1978

Biochemical Journal

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Horse liver alcohol dehydrogenase specifically carboxymethylated on cysteine-46 (a ligand to the zinc in the active site) or acetimidylated on 25 of the 30 lysine residues per subunit (including residue 228) was studied. The tryptophan fluorescence of these enzymes decreased by 35 % as pH was increased, with an apparent pKa of 9.8 + 0.2, identical with that of native enzyme. Native enzyme in the presence of 30mM-imidazole, which displaces a water molecule ligated to the zinc, also had a pKa of 9.8. The ionizable group is thus neither the water molecule nor one of the modified groups. Binding of NAD+ shifted the pKa for the fluorescence transition to 7.6 with native enzyme and to 9.0 with acetimidylated enzyme, but did not shift the pKa of carboxymethylated enzyme.Binding of NAD+ and trifluoroethanol, an unreactive alcohol, gave maximal fluorescence quenching at pH 7 with all three enzymes. The acetimidylated enzyme-NAD+-trifluoroethanol complex had an apparent pKa of 5.0, but the pK of the native enzyme complex was experimentally inaccessible. The results are interpreted in terms of coupled equilibria between two different conformational states. On binding of NAD+, the modified enzymes apparently change conformation less readily than does native enzyme, but binding of alcohol can drive the change to completion.The tryptophan fluorescence of horse liver alcohol dehydrogenase (EC 1.1.1.1) depends on the state of ionization of a group (or coupled system) on the enzyme with an apparent pKa of 9.8 ± 0.2, where the fluorescence of the alkaline form is quenched by about 35 %. Binding of NAD+ shifts the apparent PKa of the quenching curve towards 7.6, and formation of the ternary complex with NAD+ and trifluoroethanol quenches maximally at neutral pH (Wolfe et al., 1977) and results in the release of one proton per active site (Shore et al., 1974). Wolfe et al. (1977) concluded that conformational changes cause the quenching, but they did not identify the ionizable groups involved. Studies on the enzyme kinetics and the binding of NAD+ previously revealed pKa values of about 9 for free enzyme and 7 for the enzyme-NAD+ complex (Dalziel, 1963;Taniguchi et al., 1967).

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Section: Discussionmentioning

confidence: 99%

pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases

Parker¹,

Hardman²,

Plapp³

et al. 1978

Biochemical Journal

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“…We have previously shown that a conformational change of liver alcohol dehydrogenase is induced by the binding of coenzyme [16,17]. The nicotinamide moiety is essential for this conformational transition since ADP-ribose, in which this group is absent, does not induce the change [4].…”

Section: Discussionmentioning

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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“…We had shown earlier [3] that crystals of the enzyme complex with ADP-Rib were isomorphous to crystals of coenzyme-free enzyme. We anticipated that the resolution of our investigation, 0.29 nm, might be too low to permit a completely unambigous orientation of the ADP-Rib molecule into the difference electron density map.…”

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confidence: 99%

“…Complexes of liver alcohol dehydrogenase with coenzyme invariably crystallize in different crystal modifications compared to the coenzyme-free crystals. When coenzyme is soaked into preformed crystals of coenzyme-free enzyme the crystals crack almost instantaneously [3]. In order to define the probable coenzyme-binding site in the enzyme molecule it was thus necessary to study the binding of suitable analogues.…”

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confidence: 99%

The Conformation of Adenosine Diphosphoribose and 8-Bromoadenosine Diphosphoribose when Bound to Liver Alcohol Dehydrogenase

et al. 1975

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8-Bromo-adenosine diphosphoribose (br'ADP-Rib) and nicotinamide 8-bromoadenine dinucleotide (Nbr'AD') which are analogues of the coenzyme NAD' , were prepared and their liver alcohol dehydrogenase complexes studied by crystallographic methods. Nbr'AD' is active in hydrogen transport and br' ADP-Rib is a coenzyme competitive inhibitor for the enzymes liver alcohol dehydrogenase and yeast alcohol dehydrogenase. X-ray data were obtained for the complex between liver alcohol dehydrogenase and br8ADP-Rib to 0.45 nm resolution and for the liver alcohol dehydrogenase-adenosine diphosphoribose complex to 0.29-nm resolution. The conformations of these analogues were determined from the X-ray data. It was found that ADP-Rib had a conformation very similar to the corresponding part of NAD', when NAD' is bound to lactate and malate dehydrogenase.br'ADP-Rib had the same anti conformation of the adenine ring with respect to the ribose as ADP-Rib and NAD', in contrast to the syn conformation Sound in 8-bromo-adenosine. The overcrowding at the 8-position is relieved in br'ADP-Rib by having the ribose in the 2' endu conformation instead of the usual 3' endo as in ADP-Rib and NAD'.

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Crystallization of Horse Liver Alcohol Dehydrogenase Complexes from Alcohol Solutions.

Cited by 61 publications

References 0 publications

pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases

pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases

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

The Conformation of Adenosine Diphosphoribose and 8-Bromoadenosine Diphosphoribose when Bound to Liver Alcohol Dehydrogenase

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