Åke Åkeson

Three-dimensional structure of horse liver alcohol dehydrogenase at 2.4 Å resolution

Abstract The crystal structure analysis of horse liver alcohol dehydrogenase has been extended to 2.4 A resolution. From the corresponding electron density map of the apoenzyme we have determined the positions of the 374 amino acids in the polypeptide chain of each subunit. The coenzyme binding domain of the subunit comprises residues 176 to 318. 45% of these residues are helical and 32% are in the central six-stranded pleated sheet structure. The positions and orientations of the helices with respect to the pleated sheet indicate a possible folding mechanism for this part of the subunit structure. The coenzyme analogue ADP-ribose binds to this domain in a position and orientation very similar to coenzyme binding to lactate dehydrogenase. The adenine part binds in a hydrophobic pocket, the adenosine ribose is hydrogen-bonded to the side chain of Asp223, the pyrophosphate is positioned by interaction with Arg47 and the nicotinamide ribose is 6A away from the catalytic zinc atom. The catalytic domain is mainly built up from three distinct antiparallel pleated-sheet regions. Residues within this domain provide ligands to the catalytic zinc atom; Cys46, His67 and Cys174. An approximate tetrahedral coordination of this zinc is completed by a water molecule or hydroxyl ion depending on the pH. Residues 95 to 113 form a lobe that binds the second zinc atom of the subunit. This zinc is liganded in a distorted tetrahedral arrangement by four sulphur atoms from the cysteine residues 97, 100, 103 and 111. The lobe forms one side of a significant cleft in the enzyme surface suggesting that this region might constitute a second catalytic centre of unknown function. The two domains of the subunit are separated by a crevice that contains a wide and deep hydrophobic pocket. The catalytic zinc atom is at the bottom of this pocket, with the zinc-bound water molecule projecting out into the pocket. This water molecule is hydrogen-bonded to the side chain of Ser48 which in turn is hydrogen-bonded to His51. The pocket which in all probability is the binding site for the substrate and the nicotinamide moiety of the coenzyme, is lined almost exclusively with hydrophobic side chains. Both subunits contribute residues to each of the two substrate binding pockets of the molecule. The only accessible polar groups in the vicinity of the catalytic centre are Ser48 and Thr178 apart from zinc and the zinc-bound water molecule.

Structure of a triclinic ternary complex of horse liver alcohol dehydrogenase at 2.9 A resolution.

Abstract The structure of a triclinic complex between liver alcohol dehydrogenase, reduced coenzyme NADH, and the inhibitor dimethylsulfoxide has been determined to 2.9 A resolution using isomorphous replacement methods. The heavy-atom positions were derived by molecular replacement methods using phase angles derived from a model of the orthorhombic apoenzyme structure previously determined to 2.4 A resolution. A model of the present holoenzyme molecule was built on a Vector General 3400 display system using the RING system of programs. This model gave a crystallographic R -value of 37.9%. There are extensive conformational differences between the protein molecules in the two forms. The conformational change involves a rotation of 7.5 ° of the catalytic domains relative to the coenzyme binding domains. A hinge region for this rotation is defined within a hydrophobic core between two helices. The internal structures of the domains are preserved with the exception of a movement of a small loop in the coenzyme binding domain. A cleft between the domains is closed by this coenzyme-induced conformational change, making the active site less accessible from solution and thus more hydrophobic. The two crystallographically independent subunits are very similar and bind both coenzyme and inhibitor in an identical way within the present limits of error. The coenzyme molecule is bound in an extended conformation with the two ends in hydrophobic crevices on opposite sides of the central pleated sheet of the coenzyme binding domain. There are hydrogen bonds to oxygen atoms of the ribose moities from Asp223, Lys228 and His51. The pyrophosphate group is in contact with the side-chains of Arg47 and Arg369. No new residues are brought into the active site compared to the apoenzyme structure. The active site zinc atom is close to the hinge region, where the smallest structural changes occur. Small differences in the co-ordination geometry of the ligands Cys46, His67 and Cysl74 are not excluded and may account for the ordered mechanism. The oxygen atom of the inhibitor dimethylsulfoxide is bound directly to zinc confirming the structural basis for the suggested mechanism of action based on studies of the apoenzyme structure.

On the inhibitory power of some further pyrazole derivatives of horse liver alcohol dehydrogenase

Abstract We found in 1969 (1) that the inhibitory power of pyrazole on LADH was greatly increased by methyl substitution in the 4-position. In this paper we have studied the effect of increasing the size of this side chain. The inhbitory power was found to increase by a factor of two for each methyl group added in a normal side chain. Some other side chains were tested. Already the 4-butyl and 4-pentyl pyrazoles were so active that for the calculation of their true inhibition constants these had to be corrected for the concentration of the enzyme (0.0005 μN). To our knowledge this never happened before.

Separation of Isozymes of horse liver alcohol dehydrogenase and purification of the enzyme by affinity chromatography on an immobilized AMP-analogue

Abstract A mixture of the two homogeneous isozymes EE and SS of horse liver alcohol dehydrogenase (alcohol: NAD + oxidoreductase, EC 1.1.1.1) was separated on a column of N 6 -(6-aminohexyl)-AMP substituted Sepharose with a linear gradient of NAD + at pH 7.5 in the presence of 1.5 mM cholic acid. SS was eluted first at 0.4 mM NAD + , and EE later at 3.4 mM NAD + . This separation is due to true biospecific adsorption since both isozymes showed no affinity to a gel to which n -hexylamine had been covalently bound. From a horse liver crude extract alcohol dehydrogenase could be purified in a one-step procedure using a pulse of 0.1 mM NAD + plus 1.1 mM pyrazole resulting in 22 times purification with a yield of 36%. The enzyme obtained was homogeneous on dodecylsulphate-gel electrophoresis and showed a specific activity of about 3.1 units/mg when measured at pH 10.0.

Subunit interaction in horse liver alcohol dehydrogenase: 35Cl nuclear magnetic resonance studies

Abstract From 35Cl NMR measurements it is shown that the initial identity of the subunits in coenzyme-free horse liver alcohol dehydrogenase (EC 1.1.1.1) disappears on addition of 1 mole of coenzyme. This experiment indicates an interaction between the protein chaines. Isobutyramide appears to affect the enzyme affinity for coenzyme binding.

Oxidation of ω-hydroxylated fatty acids and steroids by SS-isoenzyme of liver alcohol dehydrogenase

The SS-isoenzyme of alcohol dehydrogenase from horse liver was found to be active towards ω1- and ω2-hydroxylated fatty acids, an ω-hydroxylated steroid, ethanol and a 3β-hydroxysteroid. The main part of all these activities disappeared after carboxymethylation of a cysteineresidue at the active site of LADHSS. The ω-hydroxyfatty acid dehydrogenase activity of LADHSS was of similar magnitude as that of LADHEE whereas the ω-hydroxysteroid dehydrogenase activity of LADHSS was considerably higher than that of LADHEE.