Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8‐azido‐AMP

Abstract

The photoreactive AMP analog, g-azido-AMP, stimulated the activity of biodegradative threonine dehydratase of Escherichia coli in a reversible manner and, like AMP, decreased the K , for threonine. The concentrations required for half-maximal stimulation by AMP and 8-azido-AMP were 40 pM and 1.5 pM, respectively, and the maximum stimulation by 8-azido-AMP was 25% of that seen with AMP. Gel-filtration experiments revealed that 8-azido-AMP stabilized a dimeric form of the enzyme, whereas AMP promoted a tetrameric species. When present together, AMP and 8-azido-AMP showed mutual competition in influencing catalytic activity as well as the conformational state of the protein. Since the initial report [ 13 that the biodegradative threonine dehydratase of Escherichia coli, which catalyzes the pyridoxal phosphate-dependent dehydration of threonine and serine to ammonia and corresponding 2-0x0 acids, is stimulated by AMP, considerable evidence has been gathered on the effect of this nucleotide on the conformational states, catalytic mechanism, and regulation of enzyme activity by various cellular metabolites. For example, the enzyme can bind 4 mol AMP/mol protein [2], and the AMP-free enzyme exists as a protomer of M , 35000, whereas the AMP-bound form is a tetramer of M , 140000 which exhibits a 25-fold decrease in the K , for threonine [2-41. The stimulatory effect of AMP has been correlated with obligatory oligomerization of the protomeric species [4]. Furthermore, allosteric inhibition of enzyme activity [5 -71 and catabolite inactivation by covalent protein modification of the dehydratase by intermediary metabolites [7 -91 are known to be precisely regulated by subtle variations in the concentration of AMP. Nevertheless, the binding site for AMP on the enzyme molecule remains unidentified. Recently, the complete amino acid sequence of the E. coli threonine dehydratase has been determined in this laboratory from the nucleotide sequence of the cloned gene [lo, 111, and the active-site pyridoxal-phosphate-bound lysine residue involved in the dehydration reaction has been localized on the protein primary structure [ I l , 121. This report is concerned with photolabeling of the binding site of 8-azido-AMP, a photoaffinity analog of AMP. The experiments revealed that 8-azido-AMP can mimic the allosteric modifier AMP and binds to a unique site on the dehydratase molecule which most likely represents the binding region of AMP.

Divalent metal cation binding properties of human prothymosin α

“…After centrifugation, the precipitate of cell debris was discarded and the supernatant was incubated with shaking with 150 mL of Ni/nitrilotriacetic acid agarose suspension (Qiagen) at room temperature for 1 h. The resin was washed stepwise with the same buffer, the ProTa derivative was eluted with a buffer containing 10 mm Tris/HCl (pH 6.3), 6 m urea, 0.5 m NaCl, 20% glycerol, 0.5 m imidazole and precipitated with three volumes of ethanol in the presence of 0.3 m sodium acetate (pH 5.0). The pellet was dissolved in water, and the 1 mg ProTa aliquot was subjected to radiolabelling by incubating with 100 mCi [g- 32 zz, zz-Rev, zz-NES, and zz-H1 were isolated from E. coli JM109 cells freshly transformed with pQEzz60, pQEzzRev, pQEzzNES, and pQEzzH1, respectively. For production of zz-Rev, zz-NES and zz-H1, cell cultures were grown at 30 8C in 10 mL of LB medium containing 0.2% glucose and 100 mg´mL 21 ampicillin, whereas`Super medium' (2.5% Bacto tryptone, 1.5% Bacto yeast extract, 0.5% NaCl) with the same additives was used for production of zz.…”

Macromolecular Translocation Inhibitor II (Zn2+-binding Protein, Parathymosin) Interacts with the Glucocorticoid Receptor and Enhances Transcription in Vivo

“…The localization of MTI-II, however, remains somewhat complicated. As described above, MTI-II contains a bipartite NLS and is actively translocated into the nuclei of undifferentiated cells (13,(15)(16)(17)(18), but it remains in the cytoplasm of differentiated and nonproliferated rat cells (13,15). Here, we wanted to identify the localization of MTI-II in glucocorticoid hormone-depleted cells and to determine whether MTI-II can colocalize with GR in the nucleus in the presence of hormone.…”

“…Although the N-terminal region of MTI-II consists of 28 amino acids that are homologous to thymosin ␣1 (corresponding to 46% identity), the proposed role of this protein as the precursor of thymosin ␣1 has been brought into question, because MTI-II was found to have no coding sequence for signal peptides, as would be expected for a secretory protein, but instead was found to have an active bipartite nuclear localization signal (NLS) in its C-terminal region (15). Thus, this protein is actively translocated into the nuclei of Xenopus oocytes (16), HeLa S3 cells (17), dedifferentiated rat primary hepatocytes (13), COS cells (15), Reuber H35 rat hepatoma cells (15), and T24 (human bladder carcinoma) cells (18). Furthermore, MTI-II has been reported to bind to the linker histone H1 in vitro (19).…”

“…This indicates that MTI-II has functions not only in the nucleus but also in the cytoplasm. Brand et al (13) showed that MTI-II is actively translocated into the nuclei of undifferentiated cells, whereas it stays in the cytoplasm of differentiated and nonproliferated cells of rat tissues. Further detailed studies have suggested that the nuclear/cytoplasmic distribution of the protein is regulated by cell density and is related to the differentiation state of the cell (15,24).…”

Characterization of S1 nuclease

“…The role of zinc in catalysis, structure and regulation, has been demonstrated in many enzymes (Vallee and Galdes, 1984). Recently, it has been shown that in the case or a 1PkDa protein from rat liver, which inactivates rat liver 6-phosphofructokinase, carboxylate groups serve as an excellent ligand for zinc co-ordination (Brand et al, 1988). In addition, carboxylic acid residues have also been implicated in the catalytic activity of RNAase T1 (Takahashi and Moore, 1982) and Staphylococcal nuclease (Weber et al, 1991).…”