Dinucleotide Fold Research Articles

Inhibition of monkey liver serine hydroxymethyltransferase by Cibacron Blue 3G-A.

Cibacron Blue 3G-A inhibited monkey liver serine hydroxymethyltransferase competitively with respect to tetrahydrofolate and non-competitively with respect to L-serine. NADH, a positive heterotropic effector, failed to protect the enzymes against inhibition by the dye and was unable to desorb the enzyme from Blue Sepharose CL-6B gel matrix. The binding of the dye to the free enzyme was confirmed by changes in the dye absorption spectrum. The results indicate that the dye probably binds at the tetrahydrofolate-binding domain of the enzyme, rather than at the 'dinucleotide fold'.

On the nucleotide binding domain of fructose-1,6-bisphosphatase

Abstract Native chicken liver fructose-1,6-bisphosphatase (Fru-P2ase) can bind to blue dextranSepharose affinity column and is not displaced by its sugar-phosphate substrate; however; it is readily eluted by the inhibitor 5′-AMP. Treatment of Fru-P2ase with pyridoxal 5′-phosphate (pyridoxal-P) in the presence of the substrate, fructose 1,6-bisphosphate, followed by reduction with NaBH4 leads to the formation of active pyridoxal-P derivatives of the enzyme showing diminished sensitivity to AMP inhibitor. The modified enzyme does not bind to the affinity column. On the other hand, in the presence of AMP modification of Fru-P2ase with pyridoxal-P occurs at the catalytic site; this modification does not alter its binding behavior toward the dye ligand. Blue dextran can also protect Fru-P2ase against AMP inhibition, and it is a competitive desensitizer for the nucleotide ligand. The results establish that blue dextran binds specifically to the allosteric site of the enzyme, and that the structure of this site may resemble that of the dinucleotide fold in other enzymes. Like native Fru-P2ase, digestion of pyridoxal-P-Fru-P2ase (with regulatory properties altered) with subtilisin causes a severalfold increase in the catalytic activity measured at pH 9.2, without significant change in the activity at pH 7.5, and produces a peptide with 56 amino acids. The residual subunit, Mr ~ 30,000, was found to contain all of the incorporated pyridoxal-P.

Spectroscopic studies of Cibacron Blue and Congo Red bound to dehydrogenases and kinases. Evaluation of dyes as probes of the dinucleotide fold

Spectroscopic studies of Cibacron Blue and Congo Red bound to dehydrogenases and kinases. Evaluation of dyes as probes of the dinucleotide fold

Dinucleotide fold proteins: Interaction of arabinose binding protein with Cibacron Blue 3G-A

Cibacron Blue 3G-A, the dye moiety of Blue dextran-Sepharose, has been shown to not specifically bind a protein with a dinucleotide fold-like supersecondary structure, L-arabinose binding protein from Escherichia coli. This shows that Cibacron Blue 3G-A is not suitable as a definitive probe for the dinucleotide fold as suggested earlier ( Thompson et al., 1975 ; Stellwagen, 1977). An explanation for the large predominance of proteins containing this protein supersecondary structure that bind to this dye is presented.

Binding of Cibacron Blue F3GA to Flavocytochrome b2 from Baker's Yeast

1. Flavin-free cytochrome b2 has been prepared by rapid Sephadex filtration at acid pH. The method, which yields an apo-enzyme with high reconstitution potential and has several advantages over previously used procedures, is described in detail. 2. Flavin-free cytochrome b2 thus prepared is retained by blue-dextran-bound Sepharose. It can be eluted by an increase in ionic strength, by dilute ethylene glycol and specifically by low concentrations of FMN. The holoenzyme is not retarded at all. 3. Both flavin-free and holocytochrome b2 bind Cibacron blue F3GA with appearance of distinct difference spectra. Cibacron blue is an inhibitor for the holoenzyme, it shows mixed type inhibition with respect to lactate. 4. It is concluded that there are two types of binding sites for Cibacron blue F3GA on flavocytochrome b2. Both possess ionic and hydrophobic character; one of them, which is the flavin binding site, is only available in the absence of the cofactor. Taken together these results may mean that the enzyme possesses a local flavin-binding structure similar to the 'dinucleotide fold'.

Inhibition of chicken erythrocyte AMP deaminase by tetraiodofluorescein compounds

A kinetic study has been performed on the inhibition of the chicken erythrocyte AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) reaction by tetraiodofluorescein and Rose Bengal. These dyes inhibited the enzyme by decreasing its affinity for the substrate without affecting the maximum volocity. Kinetic analysis has shown the inhibition constants for tetraiodofluorescein and Rose Bengal to be 350 and 55 μM, respectively, and the presence of 4 binding sites of the enzyme for the inhibitors per enzyme molecule. These results suggest that the fluorescein dyes mimic the AMP binding at the catalytic center for the enzyme, which can be formed by the “dinucleotide fold”.

Rapid purification and properties of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae

A method for the purification of yeast K+-activated aldehyde dehydrogenase is presented which can be completed in substantially less time than other published procedures. The enzyme has a different N-terminal amino acid from preparations previously reported, and other small differences in amino acid content. These differences may be the result of differential proteolytic digestion rather than a different protein in vivo. A purification step involves the biospecific adsorption on affinity columns containing immobilized nucleotides in the absence of the substrate aldehyde. Direct binding studies with the coenzyme in the absence of aldehyde reveal 4 NAD sites per tetrameric molecule, each with a dissociation constant of 120 micron. These results conflict with properties of preparations previously reported and may conflict with kinetic models that have aldehyde as the leading substrate. Binding to Blue Dextran affinity columns suggests the presence of a dinucleotide fold in common with other dehydrogenases and kinases.

Interaction of cibacron blue 3G-A and related dyes with nucleotide-requiring enzymes

Cibacron Blue 3G-A (I), the chromophore in Blue Dextran, its structural isomer Cibacron Brilliant Blue BR-P (II), and two other structural analogs (III, IV) were used to probe the nucleotide binding sites of selected kinases and dehydrogenases. Inhibition studies indicate that the portion of the dye molecule necessary for effective inhibition of nucleotide binding is a structure similar to 1-amino-4(4′-aminophenylamino)-anthraquinone-2,3′-disulfonic acid (ASSO; III). The strong inhibition exhibited by these dyes is likely to be due to interaction with specific nucleotide binding sites, irrespective of the presence of a “dinucleotide fold” in the proteins' supersecondary structure.

MOLECULAR CHARACTERIZATION OF CHOLINE ACETYLTRANSFERASE FROM BOVINE BRAIN CAUDATE NUCLEUS AND SOME IMMUNOLOGICAL PROPERTIES OF THE HIGHLY PURIFIED ENZYME

Abstract—Choline acetyltransferase from bovine brain caudate nucleus has been purified to a specific activity of 25–30 μmol ACh formed per min and mg protein. Disc electrophoresis at pH 9.5 of the purified enzyme showed two protein bands localized close to each other. We were not able to show if ChAT was linked to one or both bands. In SDS disc electrophoresis the enzyme preparation showed one major and one minor protein band with molecular weights of 69,000 and 34,000, respectively. Heterogeneity of the enzyme preparation was also demonstrated by immunodiffusion and immunoelectrophoresis. After ammonium sulphate precipitation no aggregation of the enzyme could be detected by gelfiltration on Ultrogel AC‐34 whilst a high molecular weight fraction was occasionally observed by gelfiltration on Sephadex G‐200. The enzyme was, however, separated into two molecular forms (A and B) on CM‐Sephadex chromatography. Both molecular forms had the same S220w but differed in heat stability and affinity for acetyl‐CoA. Both forms were inactivated by an antibody preparation raised against either a purified preparation of ChAT, or A and B separately. The highly purified enzyme preparation was inactivated more than 98% by immunoprecipitation. The antibody crossreacted with ChATs from several mammalian species, but only slightly with ChAT from pigeon. The results of binding studies with affinity columns, suggest that the enzyme contains a hydrophobic lobe and a dinucleotide fold, and that a free purine rather than a free ribosyl ring of acetyl‐CoA is important for the binding of the substrate to the active site. The hydrophobic lobe may be the same as the dinucleotide fold.

Applications of blue dextran and cibacron blue F3GA in purification and structural studies of nucleotide-requiring enzymes

Summary Cibacron Blue F3GA, the chromophore of Blue Dextran, interacted with all eight nucleotide-requiring enzymes that were examined. The conjugated chromophore (Blue Dextran) was more selective, interacting with enzymes including (but perhaps not restricted to) those known to possess the “dinucleotide fold”. The discriminating ability of these ligands should prove useful in elucidating the nature of nucleotide binding sites, and facilitate purification of selected nucleotide-requiring enzymes using affinity chromatography methods. Based on their comparable interactions with Cibacron Blue F3GA and weak interactions with Blue Dextran, it is suggested that neither yeast nor rat brain hexokinase contains the dinucleotide fold as a structural feature.

Binding of Cibacron blue F3GA to proteins containing the dinucleotide fold.

A simple, convenient, and sensitive spectrophotometric procedure is described for quantitative measurement of nucleoside phosphate binding sites constructed by the dinucleotide fold. The procedure involves difference spectral titration of such enzymes with the dye Cibacron blue F3GA in a spectral region remote from the intrinsic absorbance of proteins or natural ligands. The titration curves can be analyzed to determine the affinity of nucleoside phosphate binding sites for both the dye and the natural ligand over a potentially wide range of experimental conditions. The interaction of the dye with two proteins which contain the dinucleotide fold, lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) and phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), is illustrated.

Predicted distribution of NAD domain among glycolytic enzymes

X-RAY crystallographic studies have indicated that four dehydrogenases1–4, including lactate1 and glyceraldehyde phosphate4 dehydrogenase, contain a central parallel β sheet consisting of six strands flanked by four α helices. This super-secondary structure5 is called the dinucleotide fold or nicotinamide adenine dinucleotide (NAD) domain since it forms an NAD-binding site at the C terminus of the β sheet. The dinucleotide fold can be considered as two roughly identical mononucleotide domains each containing about 60 residues arranged in an alternating βαβαβ sequence and related by an approximate twofold axis5. The aromatic specificity site of subtilisin6,7 and the flavin-binding site of flavodoxin8,9 seem5 to be formed by a secondary structure very similar to a mononucleotide domain of the dehydrogenases. Crystallographic studies have shown that the structures of phosphoglycerate kinase10,11, hexokinase12, adenylate kinase13, phosphoglycerate mutase14 and triosephosphate isomerase15 also contain β sheets containing at least five strands flanked by at least three α helices. The nucleoside phosphate or, in the case of the mutase and isomerase, the sugar phosphate-binding site for each of these enzymes is either known10–12, or strongly suspected13–15 to be located at the C terminus of the β sheet. Differences occur, however, in some of these supersecondary structures relative to the NAD domain of the dehydrogenase family as regards the direction of the β strands12, the location of the binding site relative to the plane of the β sheet12, and most significantly, the sequential order or connectivity of the individual strands in the β sheet12–14. Whether these related supersecondary structures represent convergent5,12 or divergent evolutionary processes4,16, or both17, is a topic of active debate.

Blue dextran-sepharose: an affinity column for the dinucleotide fold in proteins.

A procedure is described to utilize blue dextran-Sepharose as an affinity chromatographic column specific for the super-secondary structure called the dinucleotide fold, which forms the binding sites for substrates and effectors on a wide range of proteins. The procedure can be used to identify proteins, either purified or in crude cellular extracts, that possess the dinucleotide fold and to significantly improve the purification procedures for those proteins that possess the fold.