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H Eklund

Publications and source records attributed to H Eklund.

At least 91 records · Page 5Linked to original sources
PubMed1984

Crystallization and preliminary crystallographic data of ribonucleotide reductase protein B2 from Escherichia coli.

The B2 subunit of ribonucleotide reductase from Escherichia coli has been crystallized from ammonium sulfate solutions at pH 6.0. Crystals grew as orthorhombic plates with cell dimensions a = 58 A, b = 73 A, and c = 205 A. The asymmetric unit probably contains one B2 dimer of molecular weight 2 X 43,000. The packing of molecules in the crystals is compatible with an elongated shape of the dimer. The crystals diffract to 2.5 A and are suitable for structural work.

Escherichia coli↗
PubMed1984

Correlation of exons with structural domains in alcohol dehydrogenase.

The intron/exon arrangement in the gene sequence of maize alcohol dehydrogenase has been compared to the three dimensional structure of liver alcohol dehydrogenase. The co-enzyme binding domain is separated from the catalytic domain by introns four and nine. Intron seven separates the co-enzyme binding domain into two structurally similar mononucleotide binding units. The first of these units is divided by introns five and six into three structurally similar alpha beta modules. Implications of these results for protein evolution is discussed. All splice junctions map close to or at the surface of the domains, and several of these cannot be identified by distance maps.

Alcohol Dehydrogenase↗
PubMed1984

Conformational and functional similarities between glutaredoxin and thioredoxins.

The tertiary structures of thioredoxin from Escherichia coli and bacteriophage T4 have been compared and aligned giving a common fold of 68 C alpha atoms with a root mean square difference of 2.6 A. The amino acid sequence of glutaredoxin has been aligned to those of the thioredoxins assuming that glutaredoxin has the same common fold. A model of the glutaredoxin molecule was built on a vector display using this alignment and the T4 thioredoxin tertiary structure. By comparison of the model with those of the thioredoxins, we have identified a molecular surface area on one side of the redox-active S-S bridge which we suggest is the binding area of these molecules for redox interactions with other proteins. This area comprises residues 33-34, 75-76 and 91-93 in E. coli thioredoxin; 15-16, 65-66 and 76-78 in T4 thioredoxin and 12-13, 59-60 and 69-71 in glutaredoxin. In all three molecules, this part of the surface is flat and hydrophobic. Charged groups are completely absent. In contrast, there is a cluster of charged groups on the other side of the S-S bridge which we suggest participates in the mechanisms of the redox reactions. In particular, a lysine residue close to an aromatic ring is conserved in all molecules.

Amino Acid Sequence↗
PubMed1983

Enantioselective affinity labelling of horse liver alcohol dehydrogenase. Correlation of inactivation kinetics with the three-dimensional structure of the enzyme.

Kinetic data for the inactivation of horse liver alcohol dehydrogenase with S-2-chloro-3-(imidazol-5-yl)propionate at pH8.2 were correlated with the three-dimensional structure of the enzyme. The R-2-chloro-3-(imidazol-5-yl)propionate enantiomer did not inactivate the enzyme, and the reaction is thus enantioselective. Inactivation follows an affinity-labelling mechanism where a reversible complex is formed before the irreversible alkylation and inactivation of the enzyme. A reversible complex is also formed with the non-inactivating enantiomer, and this shows that the selectivity occurs at the irreversible step. By using a computer-controlled display system, models of the two enantiomers of 2-chloro- and 2-bromo-3-(imidazol-5-yl)propionate were built into a model of the enzyme so that the imidazole moiety was liganded to the active-site metal, while the carboxylate group interacted with the general anion-binding site. The conformation of the imidazole derivatives and their orientation in the active site were adjusted to minimize unfavourable steric interactions. It was clear that alkylation of cysteine-46 could proceed with the S-enantiomer bound in this way, but not with the R-enantiomer. Model building thus agrees with the inactivation kinetics and indicates the structural origin of the enantioselectivity.

Affinity Labels↗
PubMed1983

Structure of the complex of active site metal-depleted horse liver alcohol dehydrogenase and NADH.

The complex between active site-specific metal-depleted horse liver alcohol dehydrogenase and NADH has been studied with X-ray crystallographic methods to 2.9 A resolution. The electron density maps revealed that only the catalytic zinc ions are removed, whereas the non-catalytic zinc sites ae fully occupied. A gross conformational change in the protein induced by co-enzyme binding takes place in this enzyme species despite the absence of the metal ion in the catalytic center. This circumstance is of great importance in the understanding and further analysis of the trigger mechanisms operating during the conformation transition in alcohol dehydrogenase, since the catalytic center is located at the hinge region for a domain rotation in the subunit, and the metal atom is essential for catalysis. The overall protein structure is the same as that of an NADH complex of the native zinc enzyme and the co-enzyme is bound in a similar manner. The local structural changes observed are restricted to the empty metal binding site.

Alcohol Dehydrogenase↗
PubMed1983

Structural studies of horse liver alcohol dehydrogenase: coenzyme, substrate and inhibitor binding.

Alcohol dehydrogenase from horse liver has been thoroughly investigated with crystallographic methods. Four different crystal forms of the enzyme have been solved and refined. They show that the enzyme exists in two predominant forms. The open form is found in the absence of coenzyme and has two long deep clefts cutting the enzyme in three units. In the closed form of the enzyme these clefts are closed around the coenzyme and substrate/inhibitor. Although there are large conformational changes in the enzyme, they are mainly restricted to relative movements of the separate domains. The internal structure of these domains is virtually identical in the open and closed forms. The coenzyme is the main cause of the conformational change and binds with a large number of interactions to the enzyme. About 4% of the enzyme surface is covered by the bound coenzyme. The nicotinamide ring is not bound to the active site zinc atom, but puts one surface of the ring in contact with the zinc coordinated cysteine sulphur atoms. The oxygen atom of the substrate binds directly to the zinc atom with the rest of the substrate close to the nicotinamide of the coenzyme. Large substrates extend into a 15-20 A long hydrophobic channel which opens up towards the solution. The widely used inhibitor pyrazole binds as a bridge between the zinc atom and the nicotinamide ring. Pyrazoles substituted in the 4-position are generally strong inhibitors. This can be properly related to the organization of the substrate channel of the enzyme.

Alcohol Dehydrogenase↗
PubMed1983

Crystal structures of the active site in specifically metal-depleted and cobalt-substituted horse liver alcohol dehydrogenase derivatives.

Two derivatives of horse liver alcohol dehydrogenase (LADH) in which the active site is specifically metal-depleted [H4Zn(n)2LADH] or specifically Co-substituted [Co(c)2-Zn(n)2LADH] have been studied by crystallographic methods. (In these formulae, "n" identifies the noncatalytic zinc ion and "c" identifies the catalytic metal ion.) X-ray data were collected for H4Zn(n)2LADH to 2.7-A resolution and for Co(c)2Zn(n)2LADH to 2.4-A resolution. Difference Fourier maps demonstrate clearly that the catalytic zinc ions are removed in H4Zn(n)2LADH, whereas the noncatalytic zinc ions are still present. A 2.5-A shift in the sulphur position of cysteine-46 and a slight torsion of the imidazole ring of histidine-67 are the only changes in the protein structure that could be detected when compared to the native zinc enzyme. The structure of Co(c)2Zn(n)2LADH is essentially the same as that of the native enzyme. Each cobalt ion is bound to the ligands cysteine-46, cysteine-174, and histidine-67 and to a water molecule in a distorted tetrahedral geometry. A slight change in the position of histidine-67 was found. No further structural changes could be observed in the protein.

Alcohol Dehydrogenase↗
PubMed1982

Binding of substrate in a ternary complex of horse liver alcohol dehydrogenase.

Horse liver alcohol dehydrogenase was crystallized from an equilibrium mixture containing predominantly NAD+ and p-bromobenzyl alcohol. X-ray diffractometer data to a resolution of 2.9 A were collected and used to compute electron density maps with phases calculated from the isomorphous enzyme . NADH . dimethyl sulfoxide complex, which has been refined to an R value of 25.6%. The electron density maps were readily interpreted in a graphics display system. Both subunits of the dimer bind coenzyme and alcohol in essentially the same manner; there is no evidence of asymmetry between subunits. The bromophenyl group is accommodated in a large hydrophobic pocket that has the side chain of Leu-116 rotated into a different position than in the complex with dimethyl sulfoxide. The alcohol oxygen is directly ligated to the catalytic zinc atom. The zinc is tetracoordinate and there is no room for a water molecule to make the zinc pentacoordinate. A hydrogen-bonded system formed with the hydroxyl groups of the alcohol, Ser-48 and nicotinamide ribose (2'), and the imidazole of His-51 may provide a proton relay system that links the buried alcohol to solvent. The insertion of the coenzyme's hydroxyl group into this system appears to install the catalytically active species. The observed structure has the pro-R hydrogen on C1 of the alcohol pointing away from C4 of the nicotinamide ring. This is probably a nonproductive complex that easily becomes productive by a rapid rotation of the alcohol to put the pro-R hydrogen within 3 A of C4 of the nicotinamide ring and in position for a direct transfer of hydrogen. A model of the productive complex readily explains the stereospecificity of hydride transfer observed for ethanol.

Alcohol Dehydrogenase↗
PubMed1982

Pyrazole binding in crystalline binary and ternary complexes with liver alcohol dehydrogenase.

Pyrazole is a strong inhibitor of liver alcohol dehydrogenase in combination with oxidized coenzyme NAD+. We have studied three different complexes of the inhibitor with the enzyme by using crystallographic methods: (1) the binary complex with pyrazole to 3.2-A resolution, (2) the ternary ternary complex with NAD+-4-iodopyrazole to 2.9-A resolution. Crystals of the binary complex are isomorphous to the apoenzyme, and pyrazole binds to the active-site zinc atom in a way analogous to imidazole. Crystals of the two ternary complexes are isomorphous with the ternary alcohol dehydrogenase-NADH-dimethyl sulfoxide complex. One of the nitrogen atoms of the pyrazole ring is directly bound to the active-site zinc atom with a Zn-N bond distance of 2.1A. The other nitrogen atom is 2 A from the C4 atom of the nicotinamide ring of the coenzyme. The iodine atom in 4-iodopyrazole is located in the hydrophobic substrate cleft. The effect of substitutions on the pyrazole ring are discussed in relation to the structure of the active site and substrate pocket. Pyrazole derivatives with long alkyl chains bound in the 4 position are outstanding inhibitors, and this property is related to the topography of the hydrophobic substrate cleft. The conformation of the oxidized coenzyme in the ternary complexes is essentially the same as that of the reduced coenzyme NADH in the NADH-dimethyl sulfoxide complex.

Alcohol Dehydrogenase↗
PubMed1979

Structural differences between apo- and holoenzyme of horse liver alcohol dehydrogenase.

The three-dimensional structure of a ternary complex of horse liver alcohol dehydrogenase with reduced nicotinamide adenine dinucleotide and the inhibitor dimethyl sulfoxide has been determined to 4.5 A resolution independently of the apoenzyme structure. The electron density maps of both structures have been compared. The two coenzyme binding domains which form the center of the dimer molecular have retained their conformation and orientation within the molecule whereas the catalytic domains rotate and narrow the cleft between the domains. The active site becomes shielded from the solution by a combination of this rotation, local movements of a loop from residues 53 to 57 and coenzyme and substrate binding. Both subunits bind coenzyme and inhibitor to the same extent. The nicotinamide ring of the coenzyme is positioned close to the active zinc atom and the inhibitor is bound to this zinc atom. The difference between the two crystallographically independent subunits is small. The proposed mechanisms of action for the enzyme based on the apoenzyme structure are confirmed by the present investigation.

Alcohol Oxidoreductases↗
PubMed1979

X-ray studies of the binding of Cibacron blue F3GA to liver alcohol dehydrogenase.

The binding of Cibacron F3GA to orthorhombic crystals of liver alcohol dehydrogenase has been studied to 0.37-nm resolution. Similarities in the binding of this dye were found for rings B, C and D with the binding of the coenzyme NAD+. However, ring A of the dye and the nicotinamide ribose part of the coenzyme are quite differently bound to the enzyme.

Alcohol Oxidoreductases↗
PubMed1978

Subunit conformation of yeast alcohol dehydrogenase.

The primary structure of yeast alcohol dehydrogenase has been compared to the known tertiary structure of the corresponding horse liver enzyme after proper alignment of the two proteins. Possible influences on the subunit conformations of all amino acid exchanges, which affect 75% of the positions, were examined from interactions in the x-ray model of the horse enzyme. In spite of the differences, 90 of 93 strictly internal residues are similar, 18 space-restricted glycine residues are conserved, 16 structurally compensated exchanges occur, all functionally essential residues are similar or identical, and 41 gaps in either sequence may be accommodated in the model. These results show that the general subunit conformations and enzymatic mechanisms of the two enzymes are largely identical. Four surface areas are changed, affecting a region with differing charges, a noncommon loop, a structure around the second zinc atom, and residues at the main dimer interface. Although the subunit interactions in the yeast enzyme cannot be determined, the surface changes probably correlate with differences in quaternary structure between the proteins.

Alcohol Oxidoreductases↗
PubMed1977

Coenzyme-induced conformational changes and substrate binding in liver alcohol dehydrogenase.

The apoenzyme and holoenzyme structures of liver alcohol dehydrogenase have been determined by X-ray methods to obtain details about coenzyme binding, substrate specificity and the catalytic mechanism. Coenzyme binding induces a conformational change of the protein which partly shields the active site from the solution. The reduced coenzyme binds in an open conformation similar to that of NAD bound to malate dehydrogenase. A hydrogen bond between Thr-178 and the carboxamide group of the coenzyme is essential for proper positioning of the nicotinamide in the active site. Coenzyme analogues in which the carboxamide group is absent or substituted with iodine bind in a different conformation and do not induce the structural change of the protein. Binding of substrate molecules has been studied in crystals obtained from an equilibrium mixture of enzyme, coenzyme and p-bromobenzyl alcohol. The oxygen atom of this substrate as well as that of the inhibitor molecules trifluoroethanol and dimethyl sulphoxide bind directly to the catalytic zinc atom. The substrate-binding region is a deep hydrophobic pocket at the bottom of which the zinc atom mediates electrophilic catalysis of alcohol oxidation.

Alcohol Oxidoreductases↗